Neuraminidase Inhibitor Compounds, Compositions and Methods for the Use Thereof in Anti-Viral Treatments

ABSTRACT

Compounds having a structure of Formula I and compositions comprising these compounds are provided. Uses of such compounds and compositions are provided for treatment or prophylaxis of viral infection. In particular, compounds and compositions may be for use in the treatment or prophylaxis of viral influenza.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/354,254, filed Jan. 19, 2012, which is a continuation-in-partapplication of U.S. application Ser. No. 13/382,284, filed Jul. 15,2010, entitled “NEURAMINIDASE INHIBITOR COMPOUNDS, COMPOSITIONS ANDMETHODS FOR THE USE THEREOF AS ANTI-VIRALS”, which application is a §371application of PCT/CA2010/001063, filed Jul. 15, 2010, which applicationclaims priority benefit of U.S. Provisional Patent Application Ser. No.61/213,786, filed Jul. 15, 2009, each of which applications isincorporated herein by reference in its entirety.

TECHNICAL FIELD

This invention relates to therapeutics, their uses and methods for thetreatment or prophylaxis of viral infection. In particular the inventionrelates to compounds, compositions, therapies, and methods of treatmentfor viral infections such as influenza.

BACKGROUND

Infection and invasion by influenza viruses requires the intermediacy ofsialic acid residues on the surface of the host cell. Sialic acid andneuraminic acid are used interchangeably. Similarly, sialidase andneuraminidase are used interchangeably. Initial attachment of the virusto the host cell occurs via the binding of the virus to these sialicacids (charged, 9-carbon sugars) through the hemagglutinin protein ofthe virus. Once inside the cell the virus replicates by taking advantageof the host cellular machinery. However, in order to remain optimallyinfective, the virus has evolved a neuraminidase that cuts off thesialic acid from the host cell surface to assist the virus in escapingthe host cell to infect other cells. Failure to cut off the sialic acidfrom the host cell surface, results in retention of virus throughattachment to the host cell.

The GH33 family of neuraminidases contains all the sialidases except theviral enzymes (GH34 family). The GH33 and GH34 families are distinctstructurally and by sequence (See Cantarel BL. et al. (2009); andHenrissat B. and Davies G J (1997) for background on Familyclassifications). Previous work has demonstrated that 2,3-difluorosialicacids are effective inhibitors of GH33 neuraminidases and that GH33neuraminidases proceed through a covalent intermediate (see for example,Watts, A. et al. (2003); Amaya, M. F. et al. (2004); Watts, A. G. andWithers, S. G. (2004); Watts, A. G. et al. (2006); Newstead, S. et al.(2008); Damager, I. et al. (2008); and Buchini, S. et al. (2008)).

The most probable mechanism for the GH34 sialidase (i.e. viralsialidases) reported in the literature is one involving an ion-pairintermediate (von Itzstein M. (2007)).

A number of compounds are known to inhibit neuraminidases. Some wellknown neuraminidase inhibitors are alkene containing sialic acidanalogues (for example: Laninamivir CAS #203120-17-6; Oseltamivir(Tamiflu) CAS #204255-11-8; and Zanamivir (Relenza) CAS #139110-80-8;see also U.S. Pat. No. 5,360,817; and Ikeda et al. Bioorganic &Medicinal Chemistry (2006) 14:7893-7897). Fluorinated sugar derivativeswith (reactive) fluoride leaving groups have been shown to be inhibitorsof a range of “retaining” glycosidases and function via formation ofparticularly stable glycosyl-enzyme intermediates (for example, Hagiwaraet al. (1994); and Buchini et al. (2008)). These reagents are quitespecific with respect to their target enzymes, have been shown to behighly bio-available, and even capable of crossing the blood-brainbarrier. Such inhibitors are mechanism-based in their action, making thedevelopment of resistance by viruses difficult, whereby any mutations inthe viral enzyme that reduce the inhibition must necessarily reduce theefficiency of the enzyme on the natural substrate, sialic acid andtherefore less likely to be tolerated.

SUMMARY

This invention is based in part on the fortuitous discovery thatcompounds having a covalent intermediate, as described herein, modulateneuraminidase. Specifically, compounds identified herein, showinhibition of neuraminidase, which may be useful for the treatment orprophylaxis of viral infection. In particular, the treatment orprophylaxis of influenza.

The compounds described herein may be used for in vivo or in vitroresearch uses (i.e. non-clinical) to investigate the mechanisms ofneuraminidase inhibition. Furthermore, these compounds may be usedindividually or as part of a kit for in vivo or in vitro research toinvestigate neuraminidase inhibition using recombinant proteins, viralstrains, cells maintained in culture, and/or animal models.Alternatively, the compounds described herein may be combined withcommercial packaging and/or instructions for use.

This invention is also based in part on the discovery that the compoundsdescribed herein, may also be used to modulate neuraminidase activityeither in vivo or in vitro for both research and therapeutic uses. Thecompounds may be used in an effective amount so that neuraminidaseactivity may be modulated. The neuraminidase may be viral. Theneuraminidase may be an influenza neuraminidase. In particular, thecompounds may be used to inhibit neuraminidase activity. The compoundsmodulatory activity may be used in either an in vivo or an in vitromodel for the study of viral infection. For example, influenzainfection. Furthermore, the compounds modulatory activity may be usedfor the treatment or prophylaxis of viral infection. The viral infectionmay be influenza.

Furthermore, this invention is based in part on the appreciation that3-fluoro-sialic acids compounds may be GH34 sialidase inhibitorsprovided that the compounds have a sufficient leaving group at carbon 2(position Z in Formula I) in addition to the appropriatestereochemistry, as described herein. Compounds identified herein, showinhibition of neuraminidase, which may be useful for the treatment orprophylaxis of viral infection. In particular the treatment orprophylaxis of influenza.

In accordance with one embodiment, there are provided compounds having a

structure of Formula I:

wherein

-   -   T is OH, C(O)NH₂, COOH or COOR¹,        -   wherein R¹ is a C₁₋₂₀ linear, branched or cyclic, saturated            or unsaturated, optionally substituted alkyl group,            -   wherein the optional substituent is selected from one or                more of the group including of: oxo, OH, F, Cl, Br, I,                NH₂, CN, SH, SO₃H and NO₂, and            -   wherein zero to ten backbone carbons of the optionally                substituted alkyl group may be optionally and                independently substituted with O, N or S;    -   Z is COOMe, F, Cl, Br, or OSO₂R²,        -   wherein R² is a C₁₋₂₀ linear, branched or cyclic, saturated            or unsaturated, optionally substituted alkyl group,            -   wherein the optional substituent is selected from one or                more of the group including of: oxo, OH, F, Cl, Br, I,                NH₂, CN, SH, SO₃H and NO₂, and            -   wherein zero to ten backbone carbons of the optionally                substituted alkyl group may be optionally and                independently substituted with O, N or S;    -   A is selected from the group including of: H, F, Cl, Br, OH, CN,        OR³, NO₂, SO₂R³, SR³ and COR³,        -   wherein R³ is a C₁₋₂₀ linear, branched or cyclic, saturated            or unsaturated, optionally substituted alkyl group,            -   wherein the optional substituent is selected from one or                more of the group including of: oxo, OH, F, Cl, Br, I,                NH₂, CN, SH, SO₃H and NO₂, and            -   wherein zero to ten backbone carbons of the optionally                substituted alkyl group may be optionally and                independently substituted with O, N or S;    -   D may be selected from the group including of: H, F, Cl, Br, OH,        CN, OR⁴, NO₂, SO₂R⁴, SR⁴ and COR⁴, provided A and D may be not        both H, and        -   wherein R⁴ may be a C₁₋₂₀ linear, branched or cyclic,            saturated or unsaturated, optionally substituted alkyl            group,            -   wherein the optional substituent may be selected from                one or more of the group including of: oxo, OH, F, Cl,                Br, I, NH₂, CN, SH, SO₃H and NO₂, and            -   wherein zero to ten backbone carbons of the optionally                substituted alkyl group may be optionally and                independently substituted with O, N or S;    -   collectively, A and D, optionally form an oxo group;    -   X may be selected from the group including of: N₃, NH₂, NHR⁵,        NHCH₃, NHCH₂CH₃, NHC(NH)NH₂, NHC(NH)NHR⁵, NR⁵R⁶, and        NHC(NH)N(R⁵)R⁶,        -   wherein R⁵ and R⁶ may be independently C₆H₅, CH₂C₆H₅ or a            C₁₋₈ alkyl group;    -   E may be selected from the group including of: NH₂, NHC(O)CH₃,        OR⁷, NHR⁷ and N(R⁷)(R⁸),        -   wherein R⁷ and R⁸ may be independently a C₁₋₂₀ linear,            branched or cyclic, saturated or unsaturated, optionally            substituted alkyl group,            -   wherein the optional substituent may be selected from                one or more of the group including of: oxo, OH, F, Cl,                Br, I, NH₂, CN, SH, SO₃H and NO₂, and            -   wherein zero to ten backbone carbons of the optionally                substituted alkyl group may be optionally and                independently substituted with O, N or S;    -   Q may be selected from the group including of: CH₂OH, CH₂R⁹,        CH(R⁹)(R¹⁰), C(R⁹)(R¹⁰)(R¹¹),

wherein

-   -   -   -   R⁹, R¹⁰ and R¹¹ may be independently CH₃ or CH₂CH₃, and                each of J and G may be independently selected from the                group: H, OH, OAc, OC(O)CH₃, F, Cl, Br, NO₂, CN, OR¹²,                SO₂R¹², COR¹² and SR¹²,                -   wherein R¹² may be CH₃, CH₂CH₃ or CH₂CH₂CH₃, and            -   M may be H, OH, OAc, OC(O)CH₃, NH₂, F or Cl, and            -   L may be H, OH, OAc, OC(O)R¹³ or NH₂,                -   wherein R¹³ may be a C₁₋₂₀ linear, branched or                    cyclic, saturated or unsaturated, optionally                    substituted alkyl group,                -   wherein the optional substituent may be selected                    from one or more of the group: oxo, OH, F, Cl, Br,                    I, NH₂, CN, SH, SO₃H and NO₂, and                -   wherein zero to ten backbone carbons of the                    optionally substituted alkyl group may be optionally                    and independently substituted with O, N or S.

In accordance with a further embodiment, there are provided compoundshaving a structure of formula I:

wherein

-   -   T may be C(O)NH₂, COOH or COOR¹,        -   wherein R¹ may be a C₁₋₂₀ linear, branched or cyclic,            saturated or unsaturated, optionally substituted alkyl            group,            -   wherein the optional substituent may be selected from                one or more of the group including of: oxo, OH, F, Cl,                Br, I, NH₂, CN, SH, SO₃H and NO₂, and            -   wherein zero to ten backbone carbons of the optionally                substituted alkyl group may be optionally and                independently substituted with O, N or S;    -   Z may be F, Cl, Br, or OSO₂R²,        -   wherein R² may be a C₁₋₂₀ linear, branched or cyclic,            saturated or unsaturated, optionally substituted alkyl            group,            -   wherein the optional substituent may be selected from                one or more of the group including of: oxo, OH, F, Cl,                Br, I, NH₂, CN, SH, SO₃H and NO₂, and            -   wherein zero to ten backbone carbons of the optionally                substituted alkyl group may be optionally and                independently substituted with O, N or S;    -   A may be F or Cl;    -   D may be H;    -   X may be selected from the group including of: NH₂, NHCH₃,        NHCH₂CH₃,    -   NHCH₂CH₂CH₃, NHCH₂CH₂CH₂CH₃, and NHC(NH)NH₂;    -   E may be NH₂ or NHC(O)CH₃;    -   Q may be selected from the group including of:

wherein

-   -   -   -   each of J and G may be independently selected from the                group: H, OH, OAc, OC(O)CH₃, F, Cl, Br, NO₂, CN, OR¹²,                SO₂R¹², COR¹² and SR¹²,                -   wherein R¹² may be CH₃, CH₂CH₃ or CH₂CH₂CH₃, and            -   M may be H, OH, OAc, OC(O)CH₃, NH₂, F or Cl, and            -   L may be H, OH, OAc, OC(O)R¹³ or NH₂,                -   wherein R¹³ may be a C₁₋₁₀ linear, branched or                    cyclic, saturated or unsaturated, optionally                    substituted alkyl group,                -   wherein the optional substituent may be selected                    from one or more of the group: oxo, OH, F, Cl, Br,                    I, NH₂, CN, SH, SO₃H and NO₂, and                -   wherein zero to ten backbone carbons of the                    optionally substituted alkyl group may be optionally                    and independently substituted with O, N or S.

In accordance with a further embodiment, there are provided compoundshaving a structure of formula I:

wherein

-   -   T may be C(O)NH₂, COOH or COOR¹,        -   wherein R¹ may be a C₁₋₂₀ linear, branched or cyclic,            saturated or unsaturated, optionally substituted alkyl            group,            -   wherein the optional substituent may be selected from                one or more of the group including of: oxo, OH, F, Cl,                Br, I, NH₂, CN, SH, SO₃H and NO₂, and            -   wherein zero to ten backbone carbons of the optionally                substituted alkyl group may be optionally and                independently substituted with O, N or S;    -   Z may be F or Cl;    -   A may be F or Cl;    -   D may be H;    -   X may be selected from the group including of: NH₂, NHCH₃,        NHCH₂CH₃,    -   NHCH₂CH₂CH₃, NHCH₂CH₂CH₂CH₃, and NHC(NH)NH₂;    -   E may be NH₂ or NHC(O)CH₃;    -   Q may be selected from the group including of:

wherein

-   -   -   -   each of J and G may be independently selected from the                group: H, OH, OAc, OC(O)CH₃, F, Cl, Br, NO₂, CN,            -   M may be H, OH, OAc; and            -   L may be H, OH, OAc.

In accordance with a further embodiment, there are provided compounds asdescribed herein for modulating viral neuraminidase activity. The viralneuraminidase may be a GH34 neuraminidase. The modulating of viralneuraminidase activity may be for the treatment of influenza in ananimal. The animal may be a mammal. The animal may be a human.

In accordance with a further embodiment, there are provided compounds asdescribed herein for use in the preparation of a medicament formodulating viral neuraminidase activity. The viral neuraminidase may bea GH34 neuraminidase. The modulating of viral neuraminidase activity maybe for the treatment of influenza in an animal. The animal may be amammal. The animal may be a human.

In accordance with a further embodiment, there are provided compounds asdescribed herein for modulating viral neuraminidase activity. The viralneuraminidase may be a GH34 neuraminidase. The modulating of viralneuraminidase activity may be for the treatment of influenza in ananimal. The animal may be a mammal. The animal may be a human.

In accordance with a further embodiment, there are providedpharmaceutical compositions which may include one or more compounds asdescribed herein and a pharmaceutically acceptable excipient. The viralneuraminidase may be a GH34 neuraminidase. The modulating of viralneuraminidase activity may be for the treatment of influenza in ananimal. The animal may be a mammal. The animal may be a human.

In accordance with a further embodiment, there are provided compounds orpharmaceutically acceptable salts thereof as described herein formodulating viral neuraminidase activity. The viral neuraminidase may bea GH34 neuraminidase. The modulating of viral neuraminidase activity maybe for the treatment of influenza in an animal. The animal may be amammal. The animal may be a human.

In accordance with a further embodiment, there is provided a method ofmodulating viral neuraminidase activity with one or more compoundsdescribed herein or a pharmaceutically acceptable salt thereof. Theviral neuraminidase may be a GH34 neuraminidase. The modulating of viralneuraminidase activity may be for the treatment of influenza in ananimal. The animal may be a mammal. The animal may be a human.

In accordance with a further embodiment, there is provided a commercialpackage which may contain one or more compounds described herein or apharmaceutically acceptable salt thereof or a pharmaceutical compositionthereof. The commercial package may optionally contain instructions forthe use of the compounds or pharmaceutically acceptable salt thereof orpharmaceutical composition thereof in the treatment of influenza.

T may be C(O)NH₂, COOH or COOR¹, wherein R¹ may be a C₁₋₂₀ linear,branched or cyclic, saturated or unsaturated, optionally substitutedalkyl group, wherein the optional substituent may be selected from oneor more of the group including of: oxo, OH, F, Cl, Br, I, NH₂, CN, SH,SO₃H and NO₂. Alternatively, T may be C(O)OCH₃, C(O)OCH₂CH₃,C(O)OCH₂CH₂CH₃, C(O)OCH₂CH₂CH₂CH₃, C(O)OCH₂CH₂CH₂CH₂CH₃,C(O)OCH₂CH₂CH₂CH₂CH₂CH₃, C(O)OCH₂CH₂CH₂CH₂CH₂CH₂CH₃,C(O)OCH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₃, or COOH. Alternatively, T may beC(O)OCH₃, C(O)OCH₂CH₃, C(O)OCH₂CH₂CH₃, C(O)OCH₂CH₂CH₂CH₃,C(O)OCH₂CH₂CH₂CH₂CH₃, or COOH.

A may be selected from the group including of: F, Cl, Br, OH, CN, OR³,NO₂, and COR³, wherein R³ may be a C₁₋₂₀ linear, branched or cyclic,saturated or unsaturated, optionally substituted alkyl group, whereinthe optional substituent may be selected from one or more of the groupincluding of: oxo, OH, F, Cl, Br, I, NH₂, CN, SH, SO₃H and NO₂, andwherein zero to ten backbone carbons of the optionally substituted alkylgroup may be optionally and independently substituted with O, N or S.Alternatively, A may be selected from the group including of: F, Cl, Br,OH, CN, OR³, and NO₂, wherein R³ may be a C₁₋₂₀ linear, branched orcyclic, saturated or unsaturated, optionally substituted alkyl group,wherein the optional substituent may be selected from one or more of thegroup including of: oxo, OH, F, Cl, Br, I, NH₂, CN, SH, SO₃H and NO₂,and wherein zero to ten backbone carbons of the optionally substitutedalkyl group may be optionally and independently substituted with O, N orS. Alternatively, A may be selected from the group including of: F, Cl,Br, OH, CN, and NO₂. Alternatively,A may be selected from the groupincluding of: F, Cl, Br, OR³, and NO₂, wherein R³ may be a C₁₋₂₀ linear,branched or cyclic, saturated or unsaturated, optionally substitutedalkyl group, wherein the optional substituent may be selected from oneor more of the group including of: oxo, OH, F, Cl, Br, I, NH₂, CN, SH,SO₃H and NO₂. Alternatively, A may be selected from the group includingof: F, Cl, and OR³, wherein R³ may be a C₁₋₂₀ linear, branched orcyclic, saturated or unsaturated, optionally substituted alkyl group,wherein the optional substituent may be selected from one or more of thegroup including of: oxo, OH, F, Cl, Br, I, NH₂, CN, SH, SO₃H and NO₂.Alternatively, A may be F, Cl. Alternatively, A may be F.

D may be selected from the group including of: H, F, Cl, Br, OH, CN,OR⁴, NO₂, and COR⁴, provided A and D may be not both H, and wherein R⁴may be a C₁₋₁₀ linear, branched or cyclic, saturated or unsaturated,optionally substituted alkyl group, wherein the optional substituent maybe selected from one or more of the group including of: oxo, OH, F, Cl,Br, I, NH₂, CN, SH, SO₃H and NO₂, and wherein zero to ten backbonecarbons of the optionally substituted alkyl group may be optionally andindependently substituted with O, N or S. Alternatively, D may beselected from the group including of: H, F, Cl, Br, OH, CN, OR⁴, NO₂,and COR⁴, provided A and D may be not both H, and wherein R⁴ may be aC₁₋₁₀ linear, branched or cyclic, saturated or unsaturated, optionallysubstituted alkyl group, wherein the optional substituent may beselected from one or more of the group including of: oxo, OH, F, Cl, Br,I, NH₂, CN, SH, SO₃H and NO₂. Alternatively, D may be selected from thegroup including of: H, F, Cl, Br, OH, CN, and NO₂, provided A and D maybe not both H. Alternatively, D may be selected from the group includingof: H, F, Cl, Br, OH and NO₂, provided A and D may be not both H.Alternatively, D may be selected from the group including of: H, F, Cl,Br, and OH, provided A and D may be not both H. Alternatively, D may beselected from the group including of: H, F, Cl, and Br, provided A and Dmay be not both H. Alternatively, D may be selected from the groupincluding of: H, F, and Cl, provided A and D may be not both H.Alternatively, D may be selected from the group including of: H, F, andCl, provided A and D may be not both H. Alternatively, D may be F or Cl.Alternatively, D may be H, or F, provided A and D may be not both H.Alternatively, D may be F. Alternatively, D may be H, provided A and Dmay be not both H.

X may be selected from the group including of: NH₂, NHR⁵, NHCH₃,NHCH₂CH₃, NHC(NH)NH₂, NHC(NH)NHR⁵, and NR⁵R⁶, wherein R⁵ and R⁶ may beindependently C₆H₅, CH₂C₆H₅ or a C₁₋₈ alkyl group. Alternatively, X maybe selected from the group including of: NH₂, NHR⁵, NHCH₃, NHCH₂CH₃,NHC(NH)NH₂, and NHC(NH)NHR⁵, wherein R⁵ may be C₆H₅, CH₂C₆H₅ or a C₁₋₈alkyl group. Alternatively, X may be selected from the group includingof: NH₂, NHR⁵, NHCH₃, NHCH₂CH₃, and NHC(NH)NH₂, wherein R⁵ may be C₆H₅,CH₂C₆H₅ or a C₁₋₈ alkyl group. Alternatively, X may be selected from thegroup including of: NH₂, NHR⁵, NHCH₃, NHCH₂CH₃, and NHC(NH)NH₂, whereinR⁵ may be C₆H₅, CH₂C₆H₅ or a C₁₋₈ alkyl group. Alternatively, X may beselected from the group including of: NH₂, NHR⁵, NHCH₃, NHCH₂CH₃, andNHC(NH)NH₂, wherein R⁵ may be a C₁₋₈ g alkyl group. Alternatively, X maybe selected from the group including of: NH₂, NHCH₃, and NHC(NH)NH₂.Alternatively, X may be selected from the group including of: NH₂,NHCH₂CH₃, and NHC(NH)NH₂. Alternatively, X may be NH₂ or NHC(NH)NH₂.

E may be selected from the group including of: NH₂, NHC(O)CH₃, OR⁷,NHR⁷, wherein R⁷ may be independently a C₁₋₁₀ linear, branched orcyclic, saturated or unsaturated, optionally substituted alkyl group,wherein the optional substituent may be selected from one or more of thegroup including of: oxo, OH, F, Cl, Br, I, NH₂, CN, SH, SO₃H and NO₂,and wherein zero to ten backbone carbons of the optionally substitutedalkyl group may be optionally and independently substituted with O, N orS. Alternatively, E may be selected from the group including of: NH₂,NHC(O)CH₃, OR⁷, and NHR⁷, wherein R⁷ may be independently a C₁₋₁₀linear, branched or cyclic, saturated or unsaturated, optionallysubstituted alkyl group, wherein the optional substituent may beselected from one or more of the group including of: oxo, OH, F, Cl, Br,I, NH₂, CN, SH, SO₃H and NO₂. Alternatively, E may be selected from thegroup including of: NH₂, NHC(O)CH₃, and OR⁷, wherein R⁷ may beindependently a C₁₋₁₀ linear, branched or cyclic, saturated orunsaturated, optionally substituted alkyl group, wherein the optionalsubstituent may be selected from one or more of the group including of:oxo, OH, F, Cl, Br, I, NH₂, CN, SH, SO₃H and NO₂. Alternatively, E maybe NH₂ or NHC(O)CH₃. Alternatively, E may be NHC(O)CH₃.

Q may be selected from the group including of: CH₂R⁹, CH(R⁹)(R¹⁰),C(R⁹)(R¹⁰)(R¹¹),

wherein R⁹, R¹⁰ and R¹¹ may be independently CH₃ or CH₂CH₃, and each ofJ and G may be independently selected from the group: H, OH, OAc,OC(O)CH₃, F, Cl, Br, NO₂, CN, OR¹², SO₂R¹², COR¹² and SR¹², wherein R¹²may be CH₃, CH₂CH₃ or CH₂CH₂CH₃, and M may be H, OH, OAc, OC(O)CH₃, NH₂,F or Cl, and L may be H, OH, OAc, OC(O)R¹³ or NH₂, wherein R¹³ may be aC₁₋₂₀ linear, branched or cyclic, saturated or unsaturated, optionallysubstituted alkyl group, and wherein the optional substituent may beselected from one or more of the group: oxo, OH, F, Cl, Br, I, NH₂, CN,SH, SO₃H and NO₂. Alternatively, Q may be selected from the groupincluding of:

each of J and G may be independently selected from the group: H, OH,OAc, OC(O)CH₃, F, Cl, Br, NO₂, CN, M may be H, OH, OAc, OC(O)CH₃, NH₂, For Cl, and L may be H, OH, OAc, OC(O)R¹³ or NH₂. Alternatively, Q may beselected from the group including of:

each of J and G may be independently selected from the group: H, OH,OAc, M may be H, OH, or OAc, and L may be H, OH, or OAc. Alternatively,Q may be:

each of J and G may be independently selected from the group: H, OH,OAc, M may be H, OH, or OAc, and L may be H, OH, or OAc. Alternatively,Q may be:

each of J and G may be independently selected from the group: OH, OAc, Mmay be OH, or OAc, and L may be OH, or OAc.

In accordance with a further embodiment, there is provided a method ofpreparing compound 2:

the method including: reacting a compound SAN3:

with Selectfluor in the presence of MeNO₂ /H₂O for at least 4 days toform compound 1:

and reacting compound 1 with diethylaminosulfur trifluoride (DAST),CH₂CL₂ at between −30° C.-0° C.

The method may further include: mixing compound 2 with NaOMe and MeOH;then mixing with Pd/C, H₂, and MeOH; and then mixing with LiOH, H₂O, andMeOH to form compound 4:

In accordance with a further embodiment, there is provided a method ofpreparing compound 12, the method may include mixing compound 2:

with NaOMe and MeOH; then mixing with AcOH until neutral; then mixingwith PMe₃, H₂O, and MeOH; and then reacting with compound VII:

in Et₃N, MeOH, and DMF to produce compound VIII:

then reacting compound VIII with LiOH, H₂O, and THF; and then reactingwith Pd/C, H₂, H₂O, and THF.

In accordance with a further embodiment, there are provided compoundswhich may be selected from one or more of the compounds set out inTABLES 2A and 2B.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a neuraminidase mechanism.

FIG. 2 shows inactivation of a neuraminidase by 2,3-difluorosialic acid(1).

FIGS. 3A & 3B show a time-dependent inactivation of influenza sialidase(subtype N9) by compound 4, indicated by concentration (3A), and re-plotof pseudo-first order inactivation kinetic constants (k_(i obs)) versusconcentration of compound 4 (3B).

FIGS. 4A & 4B show DFSA-4Gu Plasma levels after IV and INadministration.

FIGS. 5A, 5B, & 5C show DFSA-4Gu Trachea levels after IV and INadministration.

FIGS. 6A, 6B, & 6C show DFSA-4Gu Lung levels after IV and INadministration.

FIG. 7 shows a Zanamivir Lung levels after IV and IN administration.

FIG. 8 shows a survival plot of mice infected with HK1 H2N3 Influenza Avirus and treated with neuraminidase inhibitors.

DETAILED DESCRIPTION

As used herein, the phrase “C_(x-y) alkyl” or “C_(x)-C_(y) alkyl” isused as it is normally understood to a person of skill in the art andoften refers to a chemical entity that has a carbon skeleton or maincarbon chain comprising a number from x to y (with all individualintegers within the range included, including integers x and y) ofcarbon atoms. For example a “C₁₋₁₀ alkyl” is a chemical entity that has1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atom(s) in its carbon skeleton ormain chain.

As used herein, the term “branched” is used as it is normally understoodto a person of skill in the art and often refers to a chemical entitythat comprises a skeleton or main chain that splits off into more thanone contiguous chain. The portions of the skeleton or main chain thatsplit off in more than one direction may be linear, cyclic or anycombination thereof. Non-limiting examples of a branched alkyl aretert-butyl and isopropyl.

As used herein, the term “unbranched” is used as it is normallyunderstood to a person of skill in the art and often refers to achemical entity that comprises a skeleton or main chain that does notsplit off into more that one contiguous chain. Non-limiting examples ofunbranched alkyls are methyl, ethyl, n-propyl, and n-butyl.

As used herein, the term “substituted” is used as it is normallyunderstood to a person of skill in the art and often refers to achemical entity that has one chemical group replaced with a differentchemical group that contains one or more heteroatoms. Unless otherwisespecified, a substituted alkyl is an alkyl in which one or more hydrogenatom(s) is/are replaced with one or more atom(s) that is/are nothydrogen(s). For example, chloromethyl is a non-limiting example of asubstituted alkyl, more particularly an example of a substituted methyl.Aminoethyl is another non-limiting example of a substituted alkyl, moreparticularly it is a substituted ethyl. The functional groups describedherein may be substituted with, for example, and without limitation, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 substituents.

As used herein, the term “unsubstituted” is used as it is normallyunderstood to a person of skill in the art and often refers to achemical entity that is a hydrocarbon and/or does not contain aheteroatom. Non-limiting examples of unsubstituted alkyls includemethyl, ethyl, tert-butyl, and pentyl.

As used herein, the term “saturated” when referring to a chemical entityis used as it is normally understood to a person of skill in the art andoften refers to a chemical entity that comprises only single bonds.Non-limiting examples of saturated chemical entities include ethane,tert-butyl, and N⁺H₃.

As used herein the term “halogenated” is used as it would normally beunderstood to a person of skill in the art and refers to a moiety orchemical entity in which a hydrogen atom is replaced with a halogen atomsuch as chlorine, fluorine, iodine or bromine. For example, afluorinated side chain refers to a side chain wherein one or morehydrogen atoms is replaced with one or more fluorine atoms.

Non-limiting examples of saturated C₁-C₁₀ alkyl may include methyl,ethyl, n-propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl,t-butyl, n-pentyl, i-pentyl, sec-pentyl, t-pentyl, n-hexyl, i-hexyl,1,2-dimethylpropyl, 2-ethylpropyl, 1-methyl-2-ethylpropyl,1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1,2-triethylpropyl,1,1-dimethylbutyl, 2,2-dimethylbutyl, 2-ethylbutyl, 1,3-dimethylbutyl,2-methylpentyl, 3-methylpentyl, sec-hexyl, t-hexyl, n-heptyl, i-heptyl,sec-heptyl, t-heptyl, n-octyl, i-octyl, sec-octyl, t-octyl, n-nonyl,i-nonyl, sec-nonyl, t-nonyl, n-decyl, i-decyl, sec-decyl and t-decyl.Non-limiting examples of C₂-C₁₀ alkenyl may include vinyl, allyl,isopropenyl, 1-propene-2-yl, 1-butene-1-yl, 1-butene-2-yl,1-butene-3-yl, 2-butene-1-yl, 2-butene-2-yl, octenyl and decenyl.Non-limiting examples of C₂-C₁₀ alkynyl may include ethynyl, propynyl,butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl and decynyl.Saturated C₁-C₁₀ alkyl, C₂-C_(io) alkenyl or C₂-C₁₀ alkynyl may be, forexample, and without limitation, interrupted by one or more heteroatomswhich are independently nitrogen, sulfur or oxygen.

Non-limiting examples of the C₆-C₁₀ aryl group may include phenyl (Ph),pentalenyl, indenyl, naphthyl, and azulenyl. The C₆₋₉ aryl-C₁₋₄ alkylgroup may be, for example, and without limitation, a C₁₋₄ alkyl group asdefined anywhere above having a C₆₋₉ aryl group as defined anywhereabove as a substituent. The C₆₋₈ aryl-C₂₋₄ alkenyl group may be, forexample, and without limitation, a C₂₋₄ alkenyl as defined anywhereabove having a C₆₋₈ aryl group as defined anywhere above as asubstituent. The C₆₋₈ aryl-C₂₋₄ alkynyl group may be, for example, andwithout limitation, a C₂₋₄ alkynyl group as defined anywhere abovehaving a C₆₋₈ aryl group as defined anywhere above as a substituent.Non-limiting examples of the 4- to 10-membered non-aromatic heterocyclicgroup containing one or more heteroatoms which are independentlynitrogen, sulfur or oxygen may include pyrrolidinyl, pyrrolinyl,piperidinyl, piperazinyl, imidazolinyl, pyrazolidinyl, imidazolydinyl,morpholinyl, tetrahydropyranyl, azetidinyl, oxetanyl, oxathiolanyl,phthalimide and succinimide Non-limiting examples of the 5- to10-membered aromatic heterocyclic group containing one or moreheteroatoms which are independently nitrogen, sulfur or oxygen mayinclude pyrrolyl, pyridinyl, pyridazinyl, pyrimidinyl, pirazinyl,imidazolyl, thiazolyl and oxazolyl.

The embodiments involving the formulae as described herein include allpossible stereochemical alternatives, including those illustrated ordescribed herein.

In some embodiments, the compounds as described herein or acceptablesalts thereof above may be used for systemic treatment or prophylaxis ofa viral infection. In some embodiments, the compounds as describedherein or acceptable salts thereof above may be used in the preparationof a medicament or a composition for systemic treatment or prophylaxisof a viral infection. In some embodiments, methods of systemicallytreating any of the infections described herein are also provided. Someembodiments, make use of compositions comprising a compound describedherein and a pharmaceutically acceptable excipient or carrier. In someembodiments, the viral infection is caused, at least in part, by aninfluenza virus. Methods of treating any of the indications describedherein are also provided. Such methods may include administering acompound as described herein or a composition of a compound as describedherein, or an effective amount of a compound as described herein orcomposition of a compound as described herein to a subject in needthereof.

Compounds as described herein may be in the free form or in the form ofa salt thereof. In some embodiments, compounds as described herein maybe in the form of a pharmaceutically acceptable salt, which are known inthe art (Berge et al., J. Pharm. Sci. 1977, 66, 1). Pharmaceuticallyacceptable salt as used herein includes, for example, salts that havethe desired pharmacological activity of the parent compound (salts whichretain the biological effectiveness and/or properties of the parentcompound and which are not biologically and/or otherwise undesirable).Compounds as described herein having one or more functional groupscapable of forming a salt may be, for example, formed as apharmaceutically acceptable salt. Compounds containing one or more basicfunctional groups may be capable of forming a pharmaceuticallyacceptable salt with, for example, a pharmaceutically acceptable organicor inorganic acid. Pharmaceutically acceptable salts may be derivedfrom, for example, and without limitation, acetic acid, adipic acid,alginic acid, aspartic acid, ascorbic acid, benzoic acid,benzenesulfonic acid, butyric acid, cinnamic acid, citric acid,camphoric acid, camphorsulfonic acid, cyclopentanepropionic acid,diethylacetic acid, digluconic acid, dodecylsulfonic acid,ethanesulfonic acid, formic acid, fumaric acid, glucoheptanoic acid,gluconic acid, glycerophosphoric acid, glycolic acid, hemisulfonic acid,heptanoic acid, hexanoic acid, hydrochloric acid, hydrobromic acid,hydriodic acid, 2-hydroxyethanesulfonic acid, isonicotinic acid, lacticacid, malic acid, maleic acid, malonic acid, mandelic acid,methanesulfonic acid, 2-napthalenesulfonic acid, naphthalenedisulphonicacid, p-toluenesulfonic acid, nicotinic acid, nitric acid, oxalic acid,pamoic acid, pectinic acid, 3-phenylpropionic acid, phosphoric acid,picric acid, pimelic acid, pivalic acid, propionic acid, pyruvic acid,salicylic acid, succinic acid, sulfuric acid, sulfamic acid, tartaricacid, thiocyanic acid or undecanoic acid. Compounds containing one ormore acidic functional groups may be capable of forming pharmaceuticallyacceptable salts with a pharmaceutically acceptable base, for example,and without limitation, inorganic bases based on alkaline metals oralkaline earth metals or organic bases such as primary amine compounds,secondary amine compounds, tertiary amine compounds, quaternary aminecompounds, substituted amines, naturally occurring substituted amines,cyclic amines or basic ion-exchange resins. Pharmaceutically acceptablesalts may be derived from, for example, and without limitation, ahydroxide, carbonate, or bicarbonate of a pharmaceutically acceptablemetal cation such as ammonium, sodium, potassium, lithium, calcium,magnesium, iron, zinc, copper, manganese or aluminum, ammonia,benzathine, meglumine, methylamine, dimethylamine, trimethylamine,ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine,tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, hydrabamine, choline, betaine, ethylenediamine, glucosamine,glucamine, methylglucamine, theobromine, purines, piperazine,piperidine, procaine, N-ethylpiperidine, theobromine,tetramethylammonium compounds, tetraethylammonium compounds, pyridine,N,N-dimethylaniline, N-methylpiperidine, morpholine, N-methylmorpholine,N-ethylmorpholine, dicyclohexylamine, dibenzylamine,N,N-dibenzylphenethylamine, 1-ephenamine, N,N′-dibenzylethylenediamineor polyamine resins. In some embodiments, compounds as described hereinmay contain both acidic and basic groups and may be in the form of innersalts or zwitterions, for example, and without limitation, betaines.Salts as described herein may be prepared by conventional processesknown to a person skilled in the art, for example, and withoutlimitation, by reacting the free form with an organic acid, an inorganicacid, an organic base or an inorganic base, or by anion exchange orcation exchange from other salts. Those skilled in the art willappreciate that preparation of salts may occur in situ during isolationand/or purification of the compounds or preparation of salts may occurby separately reacting an isolated and/or purified compound.

In some embodiments, compounds and all different forms thereof (e.g.free forms, salts, polymorphs, isomeric forms) as described herein maybe in the solvent addition form, for example, solvates. Solvates containeither stoichiometric or non-stoichiometric amounts of a solvent inphysical association with the compound or salt thereof. The solvent maybe, for example, and without limitation, a pharmaceutically acceptablesolvent. For example, hydrates are formed when the solvent is water oralcoholates are formed when the solvent is an alcohol.

In some embodiments, compounds and all different forms thereof (e.g.free forms, salts, solvates, isomeric forms) as described herein mayinclude crystalline and/or amorphous forms, for example, polymorphs,pseudopolymorphs, conformational polymorphs, amorphous forms, or acombination thereof. Polymorphs include different crystal packingarrangements of the same elemental composition of a compound. Polymorphsusually have different X-ray diffraction patterns, infrared spectra,melting points, density, hardness, crystal shape, optical and electricalproperties, stability and/or solubility. Those skilled in the art willappreciate that various factors including recrystallization solvent,rate of crystallization and storage temperature may cause a singlecrystal form to dominate.

In some embodiments, compounds and all different forms thereof (e.g.free forms, salts, solvates, polymorphs) as described herein includeisomers such as geometrical isomers, optical isomers based on asymmetriccarbon, stereoisomers, tautomers, individual enantiomers, individualdiastereomers, racemates, diastereomeric mixtures and combinationsthereof, and are not limited by the description of the formulaillustrated for the sake of convenience.

In some embodiments, pharmaceutical compositions in accordance with thisinvention may comprise a salt of such a compound, preferably apharmaceutically or physiologically acceptable salt. Pharmaceuticalpreparations will typically comprise one or more carriers, excipients ordiluents acceptable for the mode of administration of the preparation,be it by injection, inhalation, topical administration, lavage, or othermodes suitable for the selected treatment. Suitable carriers, excipientsor diluents include those known in the art for use in such modes ofadministration.

Suitable pharmaceutical compositions may be formulated by means known inthe art and their mode of administration and dose determined by theskilled practitioner. For parenteral administration, a compound may bedissolved in sterile water or saline or a pharmaceutically acceptablevehicle used for administration of non-water soluble compounds such asthose used for vitamin K. For enteral administration, the compound maybe administered in a tablet, capsule or dissolved in liquid form. Thetablet or capsule may be enteric coated, or in a formulation forsustained release. Many suitable formulations are known, including,polymeric or protein microparticles encapsulating a compound to bereleased, ointments, pastes, gels, hydrogels, or solutions which can beused topically or locally to administer a compound. A sustained releasepatch or implant may be employed to provide release over a prolongedperiod of time. Many techniques known to one of skill in the art aredescribed in Remington: the Science & Practice of Pharmacy by AlfonsoGennaro, 20^(th) ed., Lippencott Williams & Wilkins, (2000).Formulations for parenteral administration may, for example, containexcipients, polyalkylene glycols such as polyethylene glycol, oils ofvegetable origin, or hydrogenated naphthalenes. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems for modulatory compounds include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes. Formulations for inhalation may contain excipients, forexample, lactose, or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may beoily solutions for administration in the form of nasal drops, or as agel. The formulations may be specifically prepared for intranasaldelivery. For example, nasal inhalation.

Compounds or pharmaceutical compositions in accordance with thisinvention or for use in this invention may be administered by means of amedical device or appliance such as an implant, graft, prosthesis,stent, etc. Also, implants may be devised which are intended to containand release such compounds or compositions. An example would be animplant made of a polymeric material adapted to release the compoundover a period of time.

An “effective amount” of a pharmaceutical composition as describedherein includes a therapeutically effective amount or a prophylacticallyeffective amount. A “therapeutically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired therapeutic result, such as reduced viral load,increased life span or increased life expectancy. A therapeuticallyeffective amount of a compound may vary according to factors such as thedisease state, age, sex, and weight of the subject, and the ability ofthe compound to elicit a desired response in the subject. Dosageregimens may be adjusted to provide the optimum therapeutic response. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the compound are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result, such as lesssevere infection or delayed or no onset, increased life span, increasedlife expectancy or prevention of the progression of infection.Typically, a prophylactic dose is used in subjects prior to or at anearlier stage of disease, so that a prophylactically effective amountmay be less than a therapeutically effective amount.

It is to be noted that dosage values may vary with the severity of thecondition to be alleviated. For any particular subject, specific dosageregimens may be adjusted over time according to the individual need andthe professional judgment of the person administering or supervising theadministration of the compositions. Dosage ranges set forth herein areexemplary only and do not limit the dosage ranges that may be selectedby medical practitioners. The amount of active compound(s) in thecomposition may vary according to factors such as the disease state,age, sex, and weight of the subject. Dosage regimens may be adjusted toprovide the optimum therapeutic response. For example, a single bolusmay be administered, several divided doses may be administered over timeor the dose may be proportionally reduced or increased as indicated bythe exigencies of the therapeutic situation. It may be advantageous toformulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage.

In some embodiments, compounds and all different forms thereof asdescribed herein may be used, for example, and without limitation, incombination with other treatment methods.

In general, compounds described herein should be used without causingsubstantial toxicity. Toxicity of the compounds of the invention can bedetermined using standard techniques, for example, by testing in cellcultures or experimental animals and determining the therapeutic index,i.e., the ratio between the LD50 (the dose lethal to 50% of thepopulation) and the LD100 (the dose lethal to 100% of the population).In some circumstances, however, such as in severe disease conditions, itmay be necessary to administer substantial excesses of the compositions.Some compounds described herein may be toxic at some concentrations.Titration studies may be used to determine toxic and non-toxicconcentrations. Toxicity may be evaluated by examining a particularcompound's or composition's specificity across cell lines. Animalstudies may be used to provide an indication if the compound has anyeffects on other tissues.

Compounds as described herein may be administered to a subject. As usedherein, a “subject” may be a human, non-human primate, rat, mouse, cow,horse, pig, sheep, goat, dog, cat, etc. The subject may be suspected ofhaving or at risk for having an infection, such as viral infection, orsuspected of having or at risk for having viral infection. Inparticular, the infection may mediated by a neuraminidase. Diagnosticmethods for viral infection, such as influenza and the clinicaldelineation of viral infection, such as influenza are known to those ofordinary skill in the art.

TABLE 1 Compounds made and tested for neuraminidase modulatory activity.

TABLE 1 2,3-Fluorinated Glycosides Compound Structure 4 (N3-100ACD)

7 (N3-102ET)

5 (N3-105ET)

9 (N3-111AMD)

12 (N3-1060U)

8 (N3-109N3)

13 (N3-106ET)

11 (N3-107AC)

23DFSA 2,3- Difluorosialic acid

C4 Bn

TABLE 2A Compounds having neuraminidase modulatory activity.

TABLE 2A 2,3-Fluorinated Glycosides with Neuraminidase ModulatoryActivity Compound Structure 4 (N3-100ACD)

7 (N3-102ET)

5 (N3-105ET)

9 (N3-111AMD)

12 (N3-106GU)

13 (N3-106ET)

C4 Bn

8

TABLE 2B Compounds made and expected to have neuraminidase modulatoryactivity.

TABLE 2B 2,3-Fluorinated Glycosides Compounds

Compounds described herein may also be used in assays and for researchpurposes.

Compounds for use in the present methods may be synthesized using themethods described herein.

Various alternative embodiments and examples are described herein. Theseembodiments and examples are illustrative and should not be construed aslimiting the scope of the invention.

Previous published work by Hagiwara et al (1994) reported3-fluoro-sialic acids as being only modest sialidase inhibitors.Specifically, they report two compounds, one with an OH group at carbon2 (position Z in Formula I). However, the OH group is not a sufficientleaving group to allow trapping of a covalent intermediate. Accordingly,the Hagiwara et al. OH compound (at C2 equivalent to Z in Formula I)showed minimal inhibition. Furthermore, the other compound tested byHagiwara et al., which has a fluorine (a sufficient leaving group) at C2(equivalent to Z in Formula I), did not have the correctstereochemistry. Accordingly, an appreciation of these requirements wasmissing in Hagiwara et al.

GENERAL METHODOLOGIES Synthesis

General methodologies for chemical preparation of compounds of Formula Iare described in the following non-limiting exemplary schemes.

Selectfluor (3.5 eq.) was added to a solution of SAN3 (1 eq.) inMeNO₂/water (3/1˜4/1) and the solution stirred for 3 days or more tocomplete the reaction at room temperature (Synthesis of SAN3-Chandler,M. et al. Journal of the Chemical Society-Perkin Transactions 1, 1995;1173-1180). The reaction mixture was quenched with saturated NaHCO₃ andextracted with EtOAc. Presence of compound (1) can easily be confirmedvia TLC. The axial-F (1) has a lower Rf value than the startingmaterial, and the equitoral-F compound and any other stereoisomers(anomeric hydroxy isomers) would have higher Rf values than the startingmaterial. The fluoride at C3 was detected at −204 ppm on ¹⁹F NMRexperiment, and the stereochemistry was assigned on the basis of ¹H- and¹⁹F-NMR coupling constants. The coupling constants J_(H3/F3) (47.9 Hz)and J_(F3/H4) (31.0 Hz) are indicative of an axial configuration of thefluorine atoms at C3, respectively.

TABLE 3 Examination for the fluorination at C3 with Selectfluor. TimeSelectivity condition (Day) Yields (axial/equitorial) 1 MeNO₂/water(3/1), rt  3 75% 5/1 2 MeNO₂/water (3/1), 60° C.  2 62% 3/1 3 DMF/water(8/1), 80° C. >30 40% 2/1 4 MeCN/water (8/1), 60° C. >30 40% 2/1

The reaction can be monitored for completion by UV on TLC, because onlythe starting material is detected under short UV. The reaction isconsidered complete upon disappearance of the UV active compound.

DAST (1.1 eq.) was added drop wise to a suspended solution of compound 1(1 eq.) in DCM −40° C. and the solution stirred vigorously for 30 min.After the mixture turned clear, the reaction mixture was quenched withsaturated NaHCO₃ and extracted with DCM and EtOAc to give compound 2. Ifthe reaction mixture doesn't turn clear after 20 min, the reaction mixis placed in a −10° C. bath for the last 10 min.

Compound 2 was hydrogenated with Pd/C in MeOH overnight at roomtemperature, the catalyst was then filtered off and 6M NaOMe was addedto the reaction mixture. The mixture was then acidified with IR120 (H+,strong) to remove the Na+. The resin was filtered off and the filtratewas evaporated and chromatographed (EtOAc/MeOH/water=15/2/1). Thehydrogenation was monitored by staining with ninhydrine solution. Bothcompounds 3 and 4 were isolated.

To a solution of compound 3 in MeCN/water (5/1) was added acetaldehyde(2 eq) at room temperature. After 30 min, 4 eq. NaCNBH₃ was added andthe reaction mixture stirred for 10 min at the same temp. The reactionmixture was quenched with 5% citric acid and chromatographed withEtOAc/acetone (9/1).

The monoacetylated amine (6) was deprotected with 6M NaOMe in wet MeOHto give compound 7.

Compound 4 was re-esterified with catalysis HCl (2M HCl in Et₂O) in dryEtOH at room temperature. The reaction mixture was left over night andthen evaporated. 2 eq. HCl was usually added. The ethyl ester can beseparated by silica gel chromatography.

The difluorinated compound 2 was deprotected with 6 M NaOMe in wet MeOHat room temperature, and compound 8 was easily purified by silica gelchromatography as 90% yields after acidification with IR120 (strong H⁺).

The azide 2 was hydrogenated with Pd/C to give compound 3, then NH₃(g)was bubbled to remove acetate and to give amide 4 with good yield.

For the preparation of compound 11, the amine 3 was acetylated with Ac₂Oin pyridine, and O-acetylates and methyl ester were selectively removedwith NaOMe. N-acetylated compound 11 was purified and obtained in 70%yield (three steps overall).

The guanylated compound 12 was prepared with3,5-dimethylpyrazole-1-carboxamidine and Et₃N in MeOH at 60° C. for 3weeks. The reaction mixture was quenched after three weeks, and 50% ofthe starting material 4 was successfully recovered. The 4-guanylated 12was isolated as 80% yield, and the ethylester 13 was given as 80% yieldafter re-esterification with catalytic HCl in EtOH at room temp for 5hours.

Compounds of Formula I may be prepared by the chemical methodologiesdescribed in the following non-limiting exemplary scheme.

Synthesis of 4-amino derivatives of 2,3-difluorosialic acid. Reagentsand conditions are as follows: Ref 1 is Chandler et al. (1995) J ChemSoc. Perk. Trans 1, 1173-1180; (a) 4 eq. Selectfluor, MeNO₂/water (3/1),rt.; (b) DAST, DCM, −40° C. (c) NaOMe, wet MeOH, rt.; (d) Pd/C, H₂,MeOH, rt.; (e) EtOH, cat. HCl, rt.; (f) Acetaldehyde for 4, Benzaldehydefor 5, NaCNBH₃, acetone, rt. and (g)3,5-dimethylpyrazole-1-carboxamidine nitrate, MeOH, 60° C.

Compounds of Formula I having an amine at C4, may alternatively beprepared by the chemical methodologies described in the followingnon-limiting exemplary scheme.

Alternatively, compounds of Formula I having a guanidine at C4, may beprepared by the chemical methodologies described in the followingnon-limiting exemplary scheme.

Alternatively, compounds of Formula I may be prepared by the chemicalmethodologies described in the following non-limiting exemplary scheme.

Alternatively, compounds of Formula I having modifications at C1, may beprepared by the chemical methodologies described in the followingnon-limiting exemplary scheme.

It will be appreciated by a person of skill in the art, that variationsin the alkyl chain length may be achieved by substituting 1-octanol (C8—having 8 carbons) for an alternative alcohol. For example, 1-octanol inthe above scheme may be substituted for an alternative primary alcohol,which may, for example, be selected from one or more of the following:Propan-1-ol (C3); Butanol (C4); 1-Pentanol (C5); 1-Hexanol (C6);1-Heptanol (C7); 1-Nonanol (C9); 1-Decanol (C10); Undecanol (C11);Dodecanol (C12); 1-Tetradecanol (C14); Cetyl alcohol (C16); Stearylalcohol (C18); and Arachidyl alcohol (C20). Similarly, it will beappreciated that an alternative substrate for this reaction may bechosen. For example, instead of DFSA-4NH2 (compound 4), compound 12(DFSA-4Gu), or compound 7, etc. may be substituted.

Alternatively, compounds of Formula I having modifications at C1, may beprepared by the chemical methodologies described in the followingnon-limiting exemplary scheme. For example, a hydrochloric acid salt ofethyl5-acetamido-4-guanyl-2,3,4,5,-tetradeoxy-3-fluoro-D-erythro-β-L-manno-non-2-ulopyranosylonatefluoride is shown below, which also adds an ethyl group at C1 (R¹). Itwill be appreciated by a person of skill in the art, that variations inthe salt produced may be achieved by substituting an alternative acidand that the length of the alkyl group at C1 may be adjusted bysubstituting an alternative alcohol as set out above.

Alternatively, compounds of Formula I having modifications at ring C6,may be prepared by the chemical methodologies described in the followingnon-limiting exemplary schemes.

It will be appreciated by a person of skill in the art, that variationsin the alkyl chain length may be achieved by substituting eitherTrimethyl orthooctyrate (C8—having 8 carbons) or Trimethyl orthobutyrate(C4—having 4 carbons) for an alternative ortho ester derivative. Forexample, Trimethyl orthooctyrate or Trimethyl orthobutyrate in the aboveschemes may be substituted for an alternative ortho ester derivative,which may, for example, be selected from one or more of the following:Trimethyl orthoacetate (C2); Trimethyl orthopropionate (C3); Trimethylorthopentionate (C5); Trimethyl orthohexanate (C6); Trimethylorthoheptanate (C7); Trimethyl orthononate (C9); Trimethyl orthodecanate(C10). Similarly, it will be appreciated that an alternative substratefor this reaction may be chosen.

Characterization(2R,3R)-4-Azido-4-deoxy-3-fluoro-7,8,9-tri-O-acetyl-sialic acid methylester (1)

ESI-MS m/z 515.1 (+Na); ¹⁹F-NMR (CFCl₃, 282 MHz) δ −204.7 (dd, J 47.9and 31.0 Hz); ¹H NMR (300 MHz) δ 5.32 (1H, m); 5.14 (1H, m); 5.00 (1H,dd, J 31.4 and 1.5 Hz), 4.86 (1H, dd, J 4.5 and 1.9 Hz), 4.25 (1H, m),4.15 (1H, m), 4.04 (1H, dd, J 12.5 and 8.5 Hz), 3.83 (1H, m), 3.77 (3H,OMe), 2.02 (12H, 4s, 4 Ac)

(2R,3R)-4-Azido-2,4-dideoxy-2,3-difluoro-7,8,9-tri-O-acetyl-sialic acidmethyl ester (2)

ESI-MS m/z=517.0 (+Na); ¹⁹F-NMR (CFCl₃, 282 MHz) δ −123.2 (1F, t, 8.46Hz), −217.2 (1F, m); ¹H-NMR (300 MHz) δ 7.23 (1H, d, NH), 5.97 (1H, m),5.37 (1H, m), 5.24 (1H, m), 5.10 (1H, dd, J 49.4 and 7.3 Hz), 4.65 (1H,dt, 28.1 and 9.4 Hz), 4.51 (1H, m), 4.27 (2H, m), 3.88 (3H, d, J 7.0 Hz,OMe), 3.39 (1H, m), 2.10 (12H, m, 4Ac). ¹³C NMR (100 MHz) δ 21.6, 21.7,21.8, 24.5, 49.1, 54.7, 57.8 (dd), 62.7, 68.5, 69.6, 71.6 (d), 78.2,87.5 (dd), 105.5 (dd), 165.0 (dd), 170.6, 171.4, 172.2, 172.5

(2R,3R)-4-Azido-2,4-dideoxy-2,3-difluorosialic acid (8)

ESI-MS m/z=353.2 (−H); F-NMR (CFCl₃, 282 MHz) δ −122.3 (1F, d, J 11.3Hz), −216.4 (1F, ddd, J 50.1, 29.2 and 11.3 Hz); ¹H-NMR (300 MHz) δ 5.30(1H, dm, J 50.2 Hz), 4.34 (1H, t, J 10.8 Hz), 4.07 (1H, dd, J29.1 and11.2 Hz), 3.90 (1H, d, J 10.5 Hz), 3.74 (2H, m), 3.50 (3H, m), 1.95 (3H,s, Ac). ¹³C NMR (100 MHz) δ 23.0, 45.9 (d), 61.5 (dd), 63.9, 68.7, 71.1,74.1 (d), 88.0 (dd), 106.5 (dd), 169.2 (dd), 175.8

(2R,3R)-4-Amino-2,4-dideoxy-2,3-difluorosialylamide (9)

ESI-MS m/z=350.1 (+Na); ¹⁹F-NMR (CFCl₃, 282 MHz) δ −121.1 (1F, d, J 8.46Hz), −219.4 (1F, m); ¹H-NMR (300 MHz) δ 5.10 (1H, dm, J 49.3 Hz), 4.20(1H, t, J 10.8 Hz), 3.96 (1H, d J 10.6 Hz), 3.73 (2H, m), 3.50 (3H, m),1.95 (3H, d, J 1.2 Hz, Ac). ¹³C NMR (100 MHz) δ 22.2, 46.1, 51.5 (dd),63.2, 67.5, 70.1, 74.1, 87.0 (dd) 105.0 (dd), 168.2 (dd), 175.2

(2R,3R)-4-(N-Acetyl) amino-2,4-dideoxy-2,3-difluorosialic acid (11)

ESI-MS m/z=415.1 (+2 Na); ¹⁹F-NMR (CFCl₃, 282 MHz) δ −121.8 (1F, d, J11.9 Hz), −214.4 (1F, m); ¹H-NMR (300 MHz) δ 5.05 (1H, d, J 50.5 Hz),4.50 (1H, m), 4.18 (1H, m), 4.05 (1H, d J 10.4 Hz), 3.82 (1H, d, J 10.3Hz), 3.75 (2H m), 3.48 (2H, m), 1.89 (3H, s, Ac), 1.84 (3H, s, Ac). ¹³CNMR (100 MHz) δ 21.6, 21.8 (d), 45.2, 50.8 (m), 63.1, 68.0, 70.5, 70.6,73.3 (d), 73.5, 169.5 (dd), 174.2, 174.8

(2R,3R)-4-(N-Ethyl) amino-2,4-dideoxy-2,3-difluorosialic acid (7)

ESI-MS m/z=355.3 (−H); ¹⁹F-NMR (CFCl₃, 282 MHz) δ 121.8 (1F, d, J 11.5Hz), −216.4 (1F, m); ¹H-NMR (300 MHz) δ 5.30 (1H, dm, J 50.2 Hz), 4.29(1H, t, J 10.7 Hz), 3.82 (1H, d J 10.6 Hz), 3.73 (2H, m), 3.52 (3H, m),3.00 (2H, m), 1.95 (3H, s, Ac), 1.10 (3H, t, J 7.3 Hz). ¹³C NMR (100MHz) δ 12.4, 23.2, 41.3, 45.2, 58.0 (dd), 63.9, 64.2 (d), 68.7, 71.2,74.2 (d), 85.5 (dd), 107.2 (dd), 170.0 (dd), 176.1

(2R,3R)-4-Amino-2,4-dideoxy-2,3-difluorosialic acid (4)

ESI-MS m/z=351.2 (+Na); ¹⁹F-NMR (CFCl₃, 282 MHz) δ −122.0 (1F, d, J 11.3Hz), −217.4 (1F, m); ¹H-NMR (400 MHz) δ 5.10 (1H, ddt, J 50.4, 10.0 and5.2 Hz), 4.16 (1H, m), 3.73 (3H m), 3.45 (3H, m), 1.91 (3H, dd, J 5.1and 1.4 Hz, Ac). ¹³C NMR (100 MHz) δ 23.1, 46.8, 53.1 (dd), 63.9, 64.1,68.5, 71.4, 74.4, 89.8 (dd), 108.5 (dd), 170.4 (dd), 176.0

Ethyl (2R,3R)-4-Amino-2,4-dideoxy-2,3-difluorosialylate (5)

ESI-MS m/z=379.1 (+Na); ¹⁹F-NMR (CFCl₃, 282 MHz) δ 122.3 (1F, d, J 5.6Hz), −219.0 (1F, m); ¹H-NMR (400 MHz) δ 5.10 (1H, ddt, J 49.2, 10.0 and2.8 Hz), 4.45 (2H, m), 4.14 (1H, m), 4.05 (1H, d J 10.4 Hz), 3.88 (3Hm), 3.66 (3H, m), 2.10 (3H, d, J 1.6 Hz, NAc), 1.30 (3H, dt, J 70.4 and7.2 Hz). ¹³C NMR (100 MHz) δ 13.4, 17.1, 22.4, 44.6 (dd), 52.4 (dd),57.7, 63.2, 65.0, 67.7 (d), 70.1 (d), 73.3 (d), 86.7 (dm), 105.1 (tm),167.0 (dm), 175.4 (d)

(2R,3R)-4-Guanyl-2,4-dideoxy-2,3-difluorosialic acid (12)

ESI-MS m/z=370.3 (−H); ¹⁹F-NMR (CFCl₃, 282 MHz) δ −121.3 (1F, d, J 14.4Hz), −214.7 (1F, m); ¹H-NMR (300 MHz) δ 5.00 (1H, dm, J50.2 Hz), 4.19(1H, t, J 8.9 Hz), 3.81 (2H, m), 3.52 (3H, m), 3.10 (1H, q, J 7.3 Hz),1.90 (3H, s, Ac), 1.12 (4H, m). ¹³C NMR (100 MHz) δ 22.3, 43.5, 46.5,55.1, 63.9, 68.9, 71.5, 74.2, 89.5 (dd), 107.0 (dd), 161.5, 170.1 (dd),175.8,

Ethyl (2R,3R)-4-Guanyl-2,4-dideoxy-2,3-difluorosialylate (13)

ESI-MS m/z=421.4 (+Na); ¹⁹F-NMR (CFCl₃, 282 MHz) δ −122.5 (1F, d, J 12.0Hz), −216.0 (1F, m); ¹H-NMR (300 MHz) δ 5.16 (1H, dm, J 49.5 Hz), 4.32(2H, m), 4.18 (2H, m), 4.03 (1H, d, J 9 Hz), 3.70 (2H, m), 3.50 (3H, m),3.09 (2H, q, J 7.1 Hz), 1.90 (3H, s, Ac), 1.23 (3H, t, J 6.2 Hz).

Hydrochloric acid salt of octyl5-acetamido-4-amino-2,3,4,5,-tetradeoxy-3-fluoro-D-erythro-β-L-manno-non-2-ulopyranosylonatefluoride

ESI-MS m/z=463.3 (M+Na); ¹H-NMR (400 MHz, CH₃OD) δ 5.37 (1H, app dt , J49.92, 5.04), 4.50-4.44 (1H, m), 4.38-4.27 (2H, m), 4.21-4.11 (2H, m),3.80-3.77 (1H, m), 3.75 (1H, dd, J 6.91, 2.30), 3.69-3.66 (1H, m),3.60-3.55 (1H, m), 2.04 (3H, s), 1.80-1.73 (2H, m), 1.42-1.28 (10, m),0.91 (3H, t, J 7.00).

Hydrochloric acid salt of ethyl5-acetamido-7,8,9-tri-O-acetyl-4-amino-2,3,4,5-tetradeoxy-3-fluoro-D-erythro-μ-L-manno-non-2-ulopyranosylonatefluoride

ESI-MS m/z=483.3 (M+H); ¹H-NMR (400 MHz, CDCl₃) δ 6.99 (1H, s),5.70-5.23 (3H, m), 5.02 (1H, s), 4.61-4.08 (6H, m), 2.28-1.23 (15H, m).

Ethyl 5-acetamido-4-amino-2,3,4,5,-tetradeoxy-3-fluoro-D-erythro-μ-L-manno-non-2-ulopyranosylonatefluoride

ESI-MS m/z=379.1 (M+Na); ¹H-NMR (400 MHz, D₂O) δ 5.18 (1H, app d, J49.34), 4.38 (2H, q, J7.16), 4.20-4.14 (1H, m), 4.01 (1H, d, J 10.51),3.87-3.76 (2H, m), 3.61 (1H, dd, J 11.95, 5.86), 3.55 (1H, d, J 9.29),3.39 (1H, dd, J 30.00, 10.96), 2.03 (3H, s), 1.32 (3H, t, J 7.16).

Hydrochloric acid salt of5-acetamido-4-amino-9-butyroyl-2,3,4,5,-tetradeoxy-3-fluoro-D-erythro-μ-L-manno-non-2-ulopyranosylonatefluoride

ESI-MS m/z=421.3 (M+Na); ¹H-NMR (400 MHz, MeOD) δ 5.34 (1H, app d, J51.62), 4.47 (1H, app t, J 10.66), 4.36 (1H, d, J 10.20), 4.16 (1H, dd,J 11.42, 6.24), 4.09-3.97 (3H, m), 3.55 (1H, d, J 9.14), 2.34 (2H, t, J7.31), 2.03 (3H, s), 1.70-1.61 (2H, m), 0.96 (3H, t, J 7.31).

Hydrochloric acid salt of5-acetamido-4-amino-9-octanoyl-2,3,4,5,-tetradeoxy-3-fluoro-D-erythro-μ-L-manno-non-2-ulopyranosylonatefluoride

ESI-MS m/z=453.3 (M−H); ¹H-NMR (400 MHz, MeOD) δ 5.35 (1H, app d, J45.84), 4.58-4.28 (2H, m), 4.23-3.88 (4H, m), 3.63-3.50 (1H, m),2.48-2.26 (2H, m), 2.15-1.94 (3H, m), 1.72-1.54 (2H, m), 1.51-1.14 (8H,m), 1.03-0.88 (3H, m).

Hydrochloric acid salt of ethyl5-acetamido-4-guanyl-2,3,4,5,-tetradeoxy-3-fluoro-D-erythro-μ-L-manno-non-2-ulopyranosylonatefluoride

ESI-MS m/z=421.4 (M+Na); ¹H-NMR (300 MHz, D₂O) 5.16 (1H, app d, J 49.5Hz), 4.32 (2H, m), 4.18 (2H, m), 4.03 (1H, d, J 9 Hz), 3.70 (2H, m),3.50 (3H, m), 3.09 (2H, q, J 7.1 Hz), 1.90 (3H, s), 1.23 (3H, t, J 6.2Hz).

Synthesis of 3′ Equatorial F Compounds

General methodologies for chemical preparation of 3′ equatorialcompounds of Formula I are described in the following non-limitingexemplary schemes. Furthermore, additional modifications to the belowexemplary schemes and alternative syntheses are known in the art.

Compounds of Formula I having modifications at C1, may be prepared bythe chemical methodologies described in the following non-limitingexemplary scheme.

It will be appreciated by a person of skill in the art, that variationsin the alkyl chain length may be achieved by substituting 1-octanol (C8—having 8 carbons) for an alternative alcohol. For example, 1-octanol inthe above scheme may be substituted for an alternative primary alcohol,which may, for example, be selected from one or more of the following:Propan-1-ol (C3); Butanol (C4); 1-Pentanol (C5); 1-Hexanol (C6);1-Heptanol (C7); 1-Nonanol (C9); 1-Decanol (C10); Undecanol (C11);Dodecanol (C12); 1-Tetradecanol (C14); Cetyl alcohol (C16); Stearylalcohol (C18); and Arachidyl alcohol (C20). Similarly, it will beappreciated that an alternative substrate for this reaction may bechosen. For example, instead of the 4NH₂ (compound 7) the 4Gu compound(compound 5), or etc. may be substituted.

Alternatively, compounds of Formula I having modifications at C1, may beprepared by the chemical methodologies described in the followingnon-limiting exemplary scheme. For example, the below exemplary schemeadds an ethyl group at C1 (R¹). It will be appreciated by a person ofskill in the art, that variations in the salt produced may be achievedby substituting an alternative acid and that the length of the alkylgroup at C1 may be adjusted by substituting an alternative alcohol asset out above.

Syntheses and Characterizations of 3′ Equatorial F Compounds

Methyl5-acetamido-7,8,9-tri-O-acetyl-4-azido-4,5-dideoxy-3μ-fluoro-D-erythro-L-gluco-nonulo-pyranosonate(2): A suspension of 1 (11.1 g, 24.3 mmol), nitromethane (95 mL), water(16 mL) and Selectfluor (34.5 g, 97.5 mmol, 4 equiv.) was stirred for 7days at room temperature. (The reaction may be monitored for completionby UV on TLC, because only the starting material is detected under shortUV. The reaction is considered complete upon disappearance of the UVactive compound.). The reaction was quenched with saturated NaHCO₃ (100mL), extracted with EtOAc (4×200 mL). The organic phase was washed withsaturated NaHCO₃ (300 mL) and brine (300 mL), dried over MgSO₄. Afterevaporation, the resulting residue was purified by flash columnchromatography (CHCl₃/Acetone/EtOAc=5/1/1) to give the desired compound2 as a white solid (2.14 g, 18%). ¹H NMR (400 MHz, CDCl₃): δ 5.73 (d,1H, J 9.2 Hz, NHAc), 5.30 (dd, 1H, J_(7,8) 6.7 Hz, H-7), 5.22 (m, 1H,H-8), 4.74 (dd, 1H, J_(3,4) 9.6 Hz, J_(H3,F3) 49.0 Hz,H-3), 4.66 (s, 1H,OH), 4.40 (dd, 1H, J_(6,7) 1.8 Hz, J _(5,6) 10.5 Hz, H-6), 4.37 (dd, 1H,J_(8,9a) 2.1 Hz, H-9a), 4.20 (m, 1H, H-4), 4.04 (dd, 1H, J_(8,9b) 6.3Hz, J_(9a,9b) 12.4 Hz, H-9b), 3.96 (s, 3H, OCH₃), 3.77 (m, 1H, H-5),2.15 (s, 3H, CH₃CO), 2.11 (s, 3H, CH₃CO), 2.04 (s, 3H, CH₃CO), 2.03 (s,3H, CH₃CO). ¹³C NMR (75 MHz, CDCl₃): δ 170.9, 170.7, 170.6 (2C), 167.8,93.3 (d, J_(C2,F3) 21.8 Hz, C-2), 89.2 (d, J_(C3,F3) 193.8 Hz, C-3),70.5, 69.6, 67.7, 62.6, 62.0 (d, J_(C4,F3) 17.2 Hz, C-4), 54.55, 49.9(d, J _(C5,F3) 6.0 Hz, C-5), 23.6, 21.2, 21.0, 20.9. ¹⁹F NMR (282 MHz,CDCl₃): δ -195.46 (s, F-2 eq). ESI-MS: 515.3 [M+Na)⁺].

Methyl5-acetamido-7,8,9-tri-O-acetyl-4-azido-2,4,5-trideoxy-2α,3β-difluoro-α-D-erythro-L-gluco-nonulopyranosonate(3): To a suspension of 2 (0.62 g, 1.3 mmol) in dry DCM (18 mL) wasadded dropwise DAST (0.18 mL, 1.4 mmol, 1.1 equiv) with stirring underN₂ at −40° C. After addition, the reaction mixture was stirred for 0.5 hat −40° C., and then gradually warmed up to −10° C. The reaction wasquenched with saturated NaHCO₃, diluted with DCM (50 mL) and washed withbrine (30 mL). The water phase was extracted again with EtOAc (2×50 mL)and washed with brine (50 mL). The combined organic phase was dried overMgSO₄. After evaporation, the resulting residue was purified by flashcolumn chromatography (DCM/Acetone=8/1) to give product 3 as a whitesolid (0.566 g, 91%). ¹H NMR (400 MHz, CDCl₃): δ 5.59 (d, 1H, J 9.0 Hz,NHAc), 5.32 (m, 1H, H-8), 5.24 (dt, 1H, J_(7,8) 8.5 Hz, H-7), 4.70 (dd,1 H, J_(5,6) 10.7 Hz, J_(6,7) 1.6 Hz, H-6), 4.66 (ddd, 1 H, J_(4,5) 10.7Hz, J_(H4,F3) 20.2 Hz, H-4), 4.47 (ddd, 1H, J_(3,4) 9.3 Hz, J_(H3,F3)48.6 Hz, J_(H3,F2) 14.5 Hz, H-3), 4.25 (dd, 1H, J_(8,9a) 2.6 Hz,H-_(9a, 4.13) (dd, 1H, J_(8,9b) 5.2 Hz, J_(9a,9b) 12.5 Hz, H-9b), 3.91(s, 3H, OCH₃), 3.62 (m, 1H, H-5), 2.14 (s, 3H, CH₃CO), 2.08 (s, 3H,CH₃CO), 2.05 (s, 3H, CH₃CO), 2.04 (s, 3H, CH₃CO). ¹³C NMR (75 MHz,CDCl₃): δ 171.0, 170.7, 170.5, 169.7, 165.3 (d, J_(C2,F2) 32.8Hz, C-1),105.5 (dd, J_(C2,F2) 229.1 Hz, J_(C2,F3) 27.2 Hz, C-2), 92.0 (dd,J_(C3,F3) 192.2 Hz, J_(C3,F2) 29.0 Hz, C-3), 72.9, 68.9, 67.0, 62.2,61.8 (dd, J_(C4,F3) 18.1 Hz, J_(C4,F2) 8.4 Hz, C-4), 53.7, 49.2 (d,J_(C5,F3) 6.9 Hz, C-5), 23.5, 21.0 (2 C), 20.9. ¹⁹F NMR (282 MHz,CDCl₃): δ −119.4 (d, J_(F2,F3) 12.7 Hz, F-2 eq), −197.5 (d, F-3 eq).ESI-MS: 517.2 [(M+Na)⁺].

Methyl 5-acetamido-7,8,9-tri-O-acetyl-4-[(N′,N″-di-tert-butoxycarbonyl)guanidine]-2,4,5-trideoxy-2α,3β-difluoro-α-D-erythro-L-gluco-nonulpyranosonate(4): A mixture of 3 (260 mg, 0.53 mmol), EtOAc (10 mL), Pd/C (10%, 60mg), N,N′-di-Boc-N″-trifluoromethanesulfonylguanidine (350 mg, 0.9 mmol,1.7 equiv) and DIPEA (0.2 mL) was placed under vacuum and then filledwith hydrogen three times, and the mixture was stirred under a H₂atmosphere for 24 h at room temperature. The reaction mixture wasfiltered through a short pad of Celite and washed with EtOAc. Afterevaporation, the resulting residue was purified by flash columnchromatography (DCM/Acetone=15/1) to give the product 4 as a white solid(0.311 g, 83%). ¹H NMR (400 MHz, CDCl₃): δ 11.37 (s, 1H, NHBoc), 8.67(d, 1H, J 7.8 Hz, NHGuanidine), 6.45 (d, 1H, J 9.0 Hz, NHAc), 5.32 (m,1H, H-8), 5.25 (brd, 1H, J_(7,8) 7.7 Hz, H-7), 4.90 (m, 1H, H-4), 4.71(ddd, 1H, J_(3,4) 8.8 Hz, J_(H3,F3) 48.5 Hz, J_(H3,F2) 12.5 Hz, H-3),4.50 (brd, 1H, H-6), 4.32 (dd, 1H, J_(8,9a) 2.6 Hz, H-9a), 4.28 (m, 1H,H-5), 4.07 (dd, 1H, J_(8,9b) 6.2 Hz, J_(9a,9b) 12.4 Hz, H-9b), 3.90 (s,3H, OCH₃), 2.15 (s, 3H, CH₃CO), 2.09 (s, 3H, CH₃CO), 2.04 (s, 3H,CH₃CO), 1.88 (s, 3H, CH₃CO), 0.99 (s, 18H, 2×Boc). ¹³C NMR (75 MHz,CDCl₃): δ 171.2, 171.1, 170.2, 169.8, 165.1 (d, J_(C2,F2) 32.8 Hz, C-1),162.7, 157.5, 152.9, 105.7 (dd, J_(C2,F2) 226.7 Hz, J_(C2,F3) 27.8 Hz,C-2), 90.4 (dd, J_(C3,F3) 191.8 Hz, J_(C3,F2) 31.9 Hz, C-3), 84.4, 80.3,74.7, 69.3, 67.2, 62.5, 53.7, 52.8 (dd, J_(C4,F3) 20.3 Hz, J_(C4,F2) 6.2Hz, C-4), 49.0 (d, J_(C5,F3) 5.0 Hz, C-5), 28.3 (3C), 28.1 93C), 23.1,21.0 (2C), 20.9. ¹⁹F NMR (282 MHz, CDCl₃): δ −115.6 (d, J_(F2,F3) 11.3Hz, F-2 eq), −195.8 (d, F-3 eq). ESI-MS: 733.4 [(M+Na)⁺].

5-Acetamido-2,4,5-trideoxy-2α,3μ-difluoro-4-guanidino-α-D-erythro-L-gluco-nonulopyranosonate(5): To a solution of 4 (71 mg, 0.1 mmol) in dry methanol (6 mL) wasadded sodium methylate solution (5.4 M, 0.1 mL) under N₂, and thereaction mixture was stirred overnight at room temperature. The reactionwas neutralized with Amberlite (IR-120), filtered and washed withmethanol, evaporated to give a residue. The resulting residue wasdissolved into TFA (1 mL) and stirred for 2 h at room temperature,evaporated and co-evaporated with toluene three times. The crude productwas purified by flash column chromatography (EtOAc/MeOH/H₂O=7/2/1) togive compound 5 as a white solid (26 mg, 92%). ¹H NMR (400 MHz, D₂O): δ4.71 (ddd, 1H, J_(3,4) 8.9 Hz, J_(H3,F3) 48.8 Hz, J_(H3,F2) 13.4 Hz,H-3), 4.56 (ddd, 1H, J_(H4,F3) 19.0 Hz, H-4), 4.49 (brd, 1H, H-6), 4.36(t, 1H, J_(4,5)=J_(5,6) 10.5 Hz, H-5), 3.84 (dd, 1H, J_(8,9a) 2.6 Hz,H-9a), 3.79 (m, 1H, H-8), 3.62 (dd, 1H, J_(8,9b) 6.0 Hz, J_(9a,9b) 11.5Hz, H-9b), 3.56 (brd, 1H, J_(7,8) 9.1 Hz, H-7). ¹³C NMR (75 MHz, D₂O): δ174.6, 169.6 (d, J_(C2,F2) 30.8 Hz, C-1), 157.6, 106.8 (dd, J_(C2,F2)222.1 Hz, J_(C2,F3) 27.8 Hz, C-2), 91.5 (dd, J_(C3,F3) 188.1 Hz,J_(C3,F2) 31.5 Hz, C-3), 73.5, 69.9, 67.9, 63.2, 55.7 (dd, J_(C4,F3)18.8 Hz, J_(C4,F2) 8.2 Hz, C-4), 48.4 (d, J_(C5,F3) 6.4 Hz, C-5), 21.9.¹⁹F NMR (282 MHz, D₂O): δ −112.7 (d, J_(F2,F3) 12.7 Hz, F-2 eq), −199.2(d, F-3 eq). ESI-MS: 369.4 [(M+H)⁻].

5-Acetamido-2,4,5-trideoxy-4-azido-2α,3β-difluoro-α-D-erythro-L-gluco-nonulo-pyranosonate(6): To a solution of 3 (50 mg, 0.1 mmol) in dry methanol (5 mL) wasadded sodium methylate solution (5.4 M, 50 μL) under N₂, and thereaction mixture was stirred for 2 h at room temperature. To thereaction mixture was added a couple drops of water, and stirred foranother an hour at room temperature. The reaction was neutralized withAmberlite (IR-120), filtered and washed with methanol, and evaporated togive a residue. The resulting residue was purified by flash columnchromatography (EtOAc/MeOH/H₂O=12/2/1) to give product 6 as a whitesolid (34 mg, 96%). ¹H NMR (400 MHz, CD₃OD): δ 4.69 (ddd, 1H, J_(H4,F3)19.7 Hz, H-4), 4.43 (ddd, 1H, J_(3,4) 9.0 Hz, J_(H3,F3) 50.0 Hz,J_(H3,F2) 13.5 Hz, H-3), 4.39 (brd, 1H, H-6), 4.11 (t, 1H,J_(4,5)=J_(5,6) 10.6 Hz, H-5), 3.79 (dd, 1H, J_(8,9a) 2.8 Hz, H-9a),3.77 (m, 1H, H-8), 3.64 (dd, 1H, J_(8,9b) 5.2 Hz, J_(9a,9b) 11.3 Hz,H-9b), 3.49 (brd, 1H, J_(7,8) 9.1 Hz, H-7). ¹³C NMR (75 MHz, CD₃OD): δ173.2, 169.0 (d, J_(C2,F2) 45.4 Hz, C-1), 106.5 (dd, J_(C2,F2) 222.6 Hz,J_(C2,F3) 28.0 Hz, C-2), 92.6 (dd, J_(C3,F3) 188.8 Hz, J_(C3,F2) 30.3Hz, C-3), 73.9, 70.4, 68.4, 63.4, 63.1 (dd, J_(C4,F3) 24.8 Hz, J_(C4,F2)8.0 Hz, C-4), 49.1 (d, J_(C5,F3) 6.3 Hz, C-5), 21.4. ¹⁹F NMR (282 MHz,CD₃OD): δ −115.7 (d, J_(F2,F3) 11.3 Hz, F-2 eq), −199.6 (d, F-3 eq).ESI-MS: 353.2 [(M+H)⁻].

5-Acetamido-2,4,5-trideoxy-4-amino-2α,3β-difluoro-α-D-erythro-L-gluco-nonulo-pyranosonate(7): A suspension of 6 (39 mg, 0.11 mmol) and Pd/C (10%, 12 mg) in drymethanol (8 mL) was vacuumed and filled with hydrogen for three times,and stirred overnight under H₂ atmosphere at room temperature. Thereaction mixture was filtered through a short pad of Celite and washedwith methanol. The organic solvent was evaporated to give a solid. Thesolid was dissolved in distilled water and filtered with MILLEX-GPfilter unit (pore size: 0.22 μm), and then lyophilized to give compound7 as a white solid (36 mg, 100%). ¹H NMR (400 MHz, D₂O): δ 4.83 (ddd,1H, J_(3,4) 9.1 Hz, J_(H3,F3) 49.6 Hz, J_(H3,F2) 13.2 Hz, H-3),4.46˜4.28 (m, 3H, H-4, H-5 & H-6), 3.84 (dd, 1H, J_(8,9a) 2.5 Hz, H-9a),3.79 (m, 1H, H-8), 3.62 (dd, 1H, J_(8,9b) 6.0 Hz, J_(9a,9b) 11.7 Hz,H-9b), 3.54 (brd, 1H, J_(7,8) 9.0 Hz, H-7). ¹³C NMR (100 MHz, D₂O): δ175.1, 169.2 (d, J_(C2,F2) 30.0 Hz, C-1), 106.4 (dd, J_(C2,F2) 222.0 Hz,J_(C2,F3) 28.0 Hz, C-2), 90.1 (dd, _(JC3,F3) 186.0 Hz, J_(C3,F2) 33.0Hz, C-3), 73.6, 69.9, 67.7, 63.2, 54.0 (dd, J_(C4,F3) 18.0 Hz, J_(C4,F2)7.0 Hz, C-4), 46.9 (d, J_(C5,F3) 6.0 Hz, C-5), 22.2. ¹⁹F NMR (282 MHz,D₂O): δ −113.6 (d, J_(F2,F3) 14.1 Hz, F-2 eq), −199.9 (d, F-3 eq).ESI-MS: 327.3 [(M−H)⁻].

Enzyme Kinetics

All experiments were carried out in 20 mM Tris/50 mM CaCl₂ buffer, pH7.6 containing 0.1% BSA. Cuvettes had a path length of 1 cm and wereused in either Cary 4000 or Cary 300 UV/visible spectrophotometerconnected to a circulating water bath. The data were analyzed using theprogram GraFit 4.0 (Erithacus software). Time-dependent inactivationswere performed by pre-incubating the enzyme at 30° C. in the presence ofseveral concentrations of inactivator. Residual enzyme activity wasdetermined at appropriate time intervals by the addition of an aliquotof the inactivation mixture to an assay solution containing 0.5 mM4-trifluoromethylumbelliferyl sialic acid (CF3MUSA). Kinetic parameterswere determined by measuring the initial linear increase in absorbanceat 385 nm. The initial rates at each time point were plotted as afunction of time to obtain time-dependent exponential decay curves fromwhich k_(i obs) could be obtained for each inactivator concentrationusing the equation:

(rate)_(t)=(rate)_(t=0) e ^((ki obs t))+offset.

The offset was used because the rates did not decay to zero. Theinactivation rate constant (k_(i)) and the reversible dissociationconstant for the inactivator (K_(d)) were determined by plottingk_(i obs) versus inactivator concentration to the equation:

k _(i obs) =k _(i) [I]/(K _(d) +[I]).

In the case [I]<<K_(d), a second-order rate constant (k_(i)/K_(d)) wasdetermined by fitting the data to the equation:

k _(i obs) =k _(i) [I]/K _(d).

Time-dependent reactivations were performed by applying the inactivatedenzyme solution (50 μl) to Amicon™ 10 K filter (Millipore™) to removeexcess inactivator. The filter was washed 5-times with 150 μL buffer at4° C. Enzyme activity was assayed at time intervals by the addition ofan aliquot of eluted enzyme to an assay solution containing 0.5 mMCF3MUSA. First-order rate constants for reactivation at each acceptorconcentration (k_(r obs)) were determined by direct fit of the activityversus time data to a first-order equation. Reactivation experimentswere attempted but no significant enzyme activity could be detected overtime, suggesting that the hydrolysis of the sialyl-enzyme intermediateis very slow. The Ki was determined by fitting the data to the equation:

K _(i) =K _(d)(k _(hyd) /k _(i)).

Cell-Based Assay of Influenza Anti-Viral Activity

Compounds were tested for antiviral activity using a cell based assay,which consists of making serial 2-fold dilutions of the antiviralcompounds (from 1:2 to 1:4096 in MegaVir medium in enough volume for thenumber of viruses tested—60 uL per virus), to which is added 100infectious units of the specific influenza virus and the preparationsare transferred to monolayers of MDCK cells in a microtitre plate. Theassay was carried out on a 96-well microtitre plate. The plate ismonitored for the development of influenza cytopathic effects from days3 to 5 post infection. Antiviral activity is determined by theinhibition of development of cytopathic effects. The highest dilution ofthe compound at which the monolayers are intact is taken as theend-point. Zanamivir was used as a positive control.

Dilution Preparations:

-   -   1. In row A on a clean 96-well microtitre plate, prepare 2-fold        serial dilutions of antiviral compounds from 1:2 to 1:4096 in        MegaVir medium in enough volume for the number of viruses tested        (60 uL per virus).    -   2. Transfer 55 uL of the 2-fold dilution series to a clean row        in the 96-well microtitre plate.    -   3. To the 55 uL dilution series, add 55 uL of diluted influenza        virus (at 100 TCID₅₀ per 25 ul). Also add virus to positive        control wells.    -   4. To the now 110 uL mixture, add 55 uL of 4× TPCK-treated        trypsin. Add trypsin also to positive and negative control        wells. Mix well.    -   5. Prepare also 2-fold serial dilutions from 1:2 to 1:256 for        the inoculating virus in MegaVir medium for back titration.

Plate Inoculation:

-   -   6. In a 96-well microtitre plate containing confluent monolayers        of MDCK cells in ˜200 uL MegaVir medium, transfer 75 uL of the        mixture to 2 respective rows as duplicates.    -   7. Transfer 50 uL of the positive control, and 25 uL of negative        controls to respective wells.    -   8. Transfer also 25 uL of the virus back titration in        duplicates.    -   9. Therefore in each well:        -   a. Samples: 25 uL compounds+25 uL virus+25 uL trypsin        -   b. Positive control: 25 uL virus+25 uL trypsin (no            compounds)        -   c. Negative control: 25 uL trypsin (no compounds or virus)        -   d. Back titration: 25 uL virus    -   10. The plates are incubated at 37C in a CO₂ incubator for 3        days, then observed for the appearance of cytopathic effects on        day 3 and day 5.

In Vivo Pharmacokinetic (PK) Profile Study Dose Administration

Intravenous (IV) injections—Mice were injected with the required volumeto administer the prescribed dose (mg/kg) to the animals based onindividual mouse weight using a 28 G needle. The injection volume was200 μL/20 g mouse. The mice were briefly restrained during IV injectionsfor approx. 1 minute. Dilation of the vein was achieved by holding theanimals under a heating lamp for a period of between 1-2 minutes.

Intra-nasal (IN) administration—Mice were anesthetized with isoflurane2% and 2 L/O2/min until the absence of a toe pinch reflex. Animals wererestrained in an upright position and using a micropipette, animals wereinstilled with 10 μL of the compounds into each nostril per 15 g of bodyweight. The mice were briefly restrained during the instillationprocedure for approx. 20 seconds and the rate of release was adjusted toallow the mice to inhale the compounds without forming bubbles. Themouse was placed back in the anesthetic chamber in an inverted positionfor an additional 2 minutes or until breathing returned to normal.

Pharmacokinetic Sampling

Mice were individually weighed and evenly distributed into groupsaccording to weight. Mice (n=20/group and n=4/time point) were injectedwith the test articles as described in the dose administration section.

Blood collection: Blood was collected at the time points indicated inthe study grouping table. For blood collection, mice were terminated byCO₂ inhalation and blood was collected by cardiac puncture. Upon lastbreath, mice were removed from inhalation chamber and approx 500-700 μLof blood was collected by cardiac puncture with a 25 G needle and placedinto the appropriate EDTA microtainer tube. Each tube was invertedseveral times to ensure even mixing of blood and EDTA to preventcoagulation. Blood samples were stored on ice until all samples werecollected for a particular time point and will then further processed togenerate plasma.

Plasma preparation: Plasma was prepared by centrifuging samples at 2500rpm for 15 minutes at 4° C. (rpm based on Beckman GH 3.8A rotor,RCF_(avg) 250×g), then pipetted off and placed into labelled vials onice and then frozen at −80° C. Samples were shipped on dry ice.

Tissue Collections: After blood collection the trachea and lung tissuewere harvested. Briefly, the ventral side of the neck was cut to exposethe thyroid and sternothyroid muscles. The muscles were gently teasedapart to expose the larynx and trachea, A hemostat was used to close offthe trachea (near the larynx) which then was cut just posterior to thelarynx and the entire trachea, bronchial tree was removed with the lungsattached. The trachea was separated from the lungs. Tissue was notrinsed in saline. Each tissue sample was transferred to an individuallabeled vial on ice and then frozen at −80° C. Samples were shipped ondry ice.

Observation of Animals

Mice were continually monitored for acute signs of toxicity for thefirst two hours following test compound administration. For groups ofmice in the last time point (7 days), mice were monitored 2× daily priorto sacrifice and tissue collection. Body weights of individual mice weremeasured every Monday, Wednesday and Friday over the course of thestudy.

Data Collection

Actual time of blood collection (time of day), body weights andbehavioural parameters as described in Experimental Design:Pharmacokinetic Sampling. The following records were collected:

Manual Randomization by body weight

Individual body weights

Observations

Comments

Actual time of blood & tissue collection

Identity of tissue samples

Reasons/findings related to any premature termination of animals

Observations of Animals

Evaluation of Drug-Induced Stress—All animals were observed postadministration, and at least once a day, more if deemed necessary,during the pre-treatment and treatment periods for mortality andmorbidity. In particular, signs of ill health were based on body weightloss, change in appetite, and behavioral changes such as altered gait,lethargy and gross manifestations of stress. When signs of severetoxicity were seen, the animals were terminated (CO₂ asphyxiation) and anecropsy was performed to assess other signs of toxicity. The followingorgans were examined: liver, gall bladder, spleen, lung, kidney, heart,intestine, lymph nodes and bladder. Any other unusual findings were alsonoted.

Moribund animals were terminated for humane reasons and the decision toterminate was at the discretion of the animal care technician and thestudy director. These findings were recorded as raw data and the time ofdeath will be logged on the following day.

In Vivo Mouse Model of Influenza Animals

A mouse model of influenza was used for this study. 6-7 week old femalemice (Balb/C-Mus musculus), Mice were given adapted HK1 influenza virusat 1250 pfu per mouse. Challenge doses were 3× LD50 determined in invivo titration studies Animals were housed in level 2 containment.

Sequence of Activities in the Study

Time Week Day Hour Activity 1 1 −2 First treatment 0 Inoculate withvirus 4 Second treatment Observe animals twice daily 2 16 Thirdtreatment 24 Fourth treatment Observe animals twice daily 3 40 Fifthtreatment 48 Sixth treatment Observe animals twice daily 4 64 Seventhtreatment 72 Eighth treatment Observe animals twice daily 5 88 Ninthtreatment 96 Tenth treatment Observe animals twice daily 6 112 Eleventhtreatment 120 Twelfth treatment Observe animals twice daily 2 7-14Observe animals daily 3 14-21 Observe animals daily

Intranasal Dose Administration

Mice were anesthetized with isoflurane 2% and 2 L/O2/min until theabsence of a toe pinch reflex Animals were restrained in an uprightposition and using a micropipette, animals were instilled with 10 μL ofthe compounds into each nostril. The mice were briefly restrained duringthe instillation procedure for approx. 20 seconds and the rate ofrelease should be adjusted to allow the mice to inhale the compoundswithout forming bubbles. The mice were placed back in the anestheticchamber in an inverted position for an additional 2 minutes or untiltheir breathing returned to normal.

Intranasal Virus Inoculation

Mice were anesthetized with isoflurane 2% and 2 L/O2/min until theabsence of a toe pinch reflex Animals were restrained in an uprightposition and using a micropipette, animals were instilled with 10 μL ofa virus preparation containing 3,000 pfu of influenza A virus, A/HK/1/68(H3N2) in to each nostril (total inoculum of 1,250 pfu per animal). Thevirus was prepared as a suspension in serum-free DMEM. The mice werebriefly restrained during the instillation procedure for approx. 20seconds and the rate of release should be adjusted to allow the mice toinhale the compounds without forming bubbles. The mice were placed backin the anesthetic chamber in an inverted position for an additional 2minutes or until breathing returned to normal.

Data Collection

The following records were collected:

Manual randomization by body weight

Individual body weights

Observations

Comments

Reasons/findings related to any premature termination of animals

Evaluation of Drug or Disease Induced Stress

All animals were observed post administration, twice a day duringtreatment periods and once every day thereafter for mortality andmorbidity. Signs of ill health included body weight loss, change inappetite, difficulty in breathing and behavioral changes such as alteredgait/posture, lethargy and gross manifestations of stress. At the signof severe illness (determined by >15% weight loss), the animals werecalled endpoint and terminated (CO₂ asphyxiation).

Moribund animals were terminated for humane reasons and the decision toterminate was at the discretion of the animal care technician and thestudy director. These findings were recorded as raw data and the time ofdeath were logged on the following day.

Cell-Based Assay of Influenza Anti-Viral Activity for 3′ Equatorial FCompounds

Compounds were tested for antiviral activity using a cell based assay,which consists of making serial 2-fold dilutions of the antiviralcompounds (from 1:2 to 1:4096 in MegaVir medium in enough volume for thenumber of viruses tested—60 uL per virus), to which is added 100infectious units of the specific influenza virus and the preparationsare transferred to monolayers of MDCK cells in a microtitre plate. Theassay was carried out on a 96-well microtitre plate. The plate ismonitored for the development of influenza cytopathic effects from days3 to 5 post infection. Antiviral activity is determined by theinhibition of development of cytopathic effects. The highest dilution ofthe compound at which the monolayers are intact is taken as theend-point. 4-G diF SA, Zanamivir, Oseltamivir, and Peramivir were usedas controls.

Dilution Preparations:

-   -   11. In row A on a clean 96-well microtitre plate, prepare 2-fold        serial dilutions of antiviral compounds from 1:2 to 1:4096 in        MegaVir medium in enough volume for the number of viruses tested        (60 uL per virus).    -   12. Transfer 55 uL of the 2-fold dilution series to a clean row        in the 96-well microtitre plate.    -   13. To the 55 uL dilution series, add 55 uL of diluted influenza        virus (at 100 TCID₅₀ per 25 ul ). Also add virus to positive        control wells.    -   14. To the now 110 uL mixture, add 55 uL of 4× TPCK-treated        trypsin. Add trypsin also to positive and negative control        wells. Mix well.    -   15. Prepare also 2-fold serial dilutions from 1:2 to 1:256 for        the inoculating virus in MegaVir medium for back titration.

Plate Inoculation:

-   -   16. In a 96-well microtitre plate containing confluent        monolayers of MDCK cells in ˜200 uL MegaVir medium, transfer 75        uL of the mixture to 2 respective rows as duplicates.    -   17. Transfer 50 uL of the positive control, and 25 uL of        negative controls to respective wells.    -   18. Transfer also 25 uL of the virus back titration in        duplicates.    -   19. Therefore in each well:        -   a. Samples: 25 uL compounds+25 uL virus+25 uL trypsin        -   b. Positive control: 25 uL virus+25 uL trypsin (no            compounds)        -   c. Negative control: 25 uL trypsin (no compounds or virus)        -   d. Back titration: 25 uL virus    -   20. The plates are incubated at 37 C in a CO₂ incubator for 3        days, then observed for the appearance of cytopathic effects on        day 3 and day 5.

Viruses for 3′ Equatorial F Compounds

The mutants were generated by producing viruses in derivatives ofZanamivir, all of which still had the 4-guanidinium group. Each of themutants have mutations at E119. E119 interactions are significant forhigh affinity binding of NAIs, but each substitution often only affectsbinding of a subset of the inhibitors. It is already known that E119Gconfers Zanamivir and peramivir resistance, but not Oseltamivir, whichis though to be due to altered interactions with the guanidinium group,and E119V confers Oseltamivir and 4-aminoNeu5Ac2en resistance, but notto Zanamivir or Peramivir.

Wild Type Viruses:

-   *A/Auckland/3/2009 (pandemic H1N1)-   *B/Florida/4/2006-   *A/Solomon Islands/3/2006 (seasonal H1N1) *provided by Biota    Holdings Limited-   G70C H1N9 wt-   E119G Fukui H3N2 wt-   sH1N1/01 H274Y-   sH1N1/08 wt-   B/Perth wt

Mutant Viruses:

-   *A/Auckland/3/2009 mutant 1 E119K-   *B/Florida/4/2006 mutant 1 E117D (E119D N2 numbering)-   *A/Solomon Islands/3/2006 mutant E119A *provided by Biota Holdings    Limited-   G70C H1N9-   Fukui H3N2 E119V-   sH1N1/01 H274Y-   sH1N1/08 H274Y-   B/Perth D197E

EXAMPLES

Further embodiments are described with reference to the following,non-limiting, examples.

Example 1 Viral Sialidase Enzyme Assays

Incubation of influenza sialidase with different concentrations of thevarious compounds resulted in time-dependent decreases in enzymeactivity, as expected for mechanism-based inhibitors, and as shown forcompound 4 in FIGS. 3A and B. FIGS. 3A and 3B show time-dependentinactivation of influenza sialidase (subtype N9) by compound 4. Theenzyme was incubated with the indicated concentrations of compound 4,and aliquots assayed with 0.5 mM CF3MUSA. Inactivation by compound 4 atindicated concentration (3A), and re-plot of pseudo-first orderinactivation kinetic constants (ki obs) versus concentration of compound4 (3B).

All 3-fluorosialyl fluorides showed excellent inactivation profilesagainst influenza sialidase at 30° C. except compound 8, and the halftime for the full inactivation of the sialidase was estimated as ca. 10mM for all amine derivatives (compounds 4, 7, 9, 11, and 12) (summary inTable 4). Interestingly, 4-aminated compound 4 and 4-guanylated compound12 showed good inactivation values (k_(i)/K_(d)=25 min⁻¹ mM⁻¹ forcompound 4 and k_(i)/K_(d)=24 min⁻¹mM⁻¹ for compound 12). Furthermore,compounds 4 and 12 showed slow reactivation in buffer solution at thesame temperature with the half time for the full reactivation of theinactivated sialidase being determined as 4.8 h for compound 4 and 6.7 hfor compound 12. Accordingly, the 3-fluorosialylenzyme intermediate verystably blocked the active site in influenza sialidase and theireffective K_(i)s were in the nanomolar range (93 nM for compound 4 and70 nM for compound 12).

Inactivation parameters for 2,3-Difluorosialic acid, (23DFSA) and the4-azide compound 8 could not be measured at 30° C. due to fasthydrolysis of sialylenzyme intermediate. Thus, the kinetic values forthese two inhibitors were measured at 4° C., and the estimatedinactivation numbers were given. The k_(i)/K_(d) value of compound23DFSA was determined as 196 min⁻¹ mM⁻¹, and 4-azide compound 8 and4-amine compound 4 showed 26-fold lower kinetic values at 4° C.(k_(i)/K_(d)=7.5 min⁻¹ mM⁻¹ for compound 8 and 7.3 min⁻¹ mM⁻¹ forcompound 4). Although the 4-amine derivatives showed lower inactivationkinetic values, these compounds were revealed as much better inhibitorsthan the 4-hydroxylated derivative (23DFSA) due to their very slowreactivation.

TABLE 4 Kinetic parameters for the reaction of influenza sialidase with3-fluorosialyl fluorides.^(a) k_(i)/K_(d) K_(d) t_(1/2(inactivation))k_(hyd) K_(i) Compound (min⁻¹mM⁻¹) (mM) (min) (min⁻¹) (nM) 23DFSA196^(b) ND^(b) ND^(b) ND^(b) ND^(b) C4 Bn 0.61 0.306 3.7 0.0025 4080  87.5^(b)  0.240^(b) 0.4^(b)  ND^(b) ND^(b)  9 0.34 1.016 1.9 ND ND 110.53 0.397 3.2 ND ND  7 4.1 0.014 11.7 ND ND  4 25 (7.3)^(b) 0.004 6.40.0024  93.2 12 24 0.009 3.1 0.0017  70.4 ^(a)All the experiments werecarried out in 20 mM TRIS/50 mM CaCl₂ buffer, pH 7.6 containing 0.1% BSAat 30° C. ^(b)The kinetic values were collected at 4° C. ND = notdetermined.

Example 2 Human Sialidase Enzyme Assays

Incubation of different concentrations of each compound with humansialidase resulted in no inactivation of the human sialidase even atvery high concentrations (10 mM) of compound demonstrating thespecificity of the compounds for influenza sialidases.

Example 3 Cytoprotection Assays with Influenza A Strains

The candidate inhibitors (compounds 4, 5, 7-9 and 11-13) were testedagainst Zanamivir and/or 2,3-DFSA for their cytopathic effect (CPE)against two influenza A strains. It is important to note that, althoughthe present Cytoprotection assay is a good qualitative indicator ofantiviral activity, the results are often variable (as note by others,Tisdale M. (2000)) and caution exercised when doing a quantitativeanalysis.

TABLE 5 The activity of compounds against influenza A/Brisbane/10/2007(H3N2) Replicate 1 Replicate 2 Cytopro- Cytopro- tective tectiveToxicity effect Relative effect Relative (ug/ Relative Compound (ng/mL)activity (ng/mL) activity mL) toxicity Zanamivir 277.4 1.0 369.9 1.0 5681.0 4 (DFSA- 6.9 40.0 9.2 40.2 3.6 160.0 4NH2) 5 (N3- 7.6 36.4 7.6 48.6125.0 4.5 105ET) 7 (N3- 122 2.3 61.0 6.1 7.8 72.7 102ET) 12 (DFSA- 55.55.0 74 5.0 0.9 640.0 4Gu) 13 (N3- 44.4 6.3 44.4 8.3 1.4 400.0 106ET) 11(N3- 1598 0.2 — — 102.3 5.6 107AC) 8 (N3- 104.3 2.7 — — 53.4 10.6 109N3)9 (N3- 117.6 2.4 — — 7.5 75.5 111AMD)

TABLE 6 The activity of compounds against influenza A/Denver/1/57 (H1N1)Cytoprotective effect Relative Compound (ng/mL) activity Zanamivir 4.31.0 4 (DFSA-4NH2) 887.8 0.005 7 (N3-102ET) 3906.3 0.001 5 (N3-105ET)488.3 0.009 12 (DFSA-4Gu) 887.8 0.005 13 (N3-106ET) 710.2 0.006 9(N3-111AMD) 3764.2 0.001 23DFSA 4261.4 0.001

TABLE 7 The activity of compounds 4 and 12 against a variety ofinfluenza A viruses and an Oseltamivir resistant strain (OsR) Antiviralcompound DFSA-4NH2 DFSA-4Gu (compound (compound Virus Zanamivir 4) 12)(100 × TCID₅₀) Trials [μM] [μM] [μM] A/Brisbane NML 1 0.834^(B)51.415^(B) 12.215^(B) (H3N2) A/Hong Kong (H3N2) 1 20.026^(B)1028.306^(B) 73.288^(B) 2 5.007^(B) 64.269^(B) 4.581^(B) 3 6.413^(B)>115.519^(C) 42.183^(C) A/Denver (H1N1) 1 <0.209^(A) <12.854^(A)<3.054^(A) A/New Caledonia 1 0.417A <12.854^(A) <3.054^(A) (H1N1)A/Brisbane NML 1 6.675^(A) 102.831^(A) 6.107^(A) (H1N1) 2 8.344^(B)411.323^(B) 3.054^(B) 3 2.138^(B) >115.519^(C) 4.474^(C) A/Brisbane 67701 <0.209^(A) 12.854^(A) <3.054^(A) (H1N1-OsR) 2 0.039^(B) 9.640^(B)0.382^(B) 3 0.013^(B) 2.410^(B) 0.382^(B) 4 0.025^(B) 3.971^(C)0.300^(C) Multiple experiments were performed on different days on somestrains ^(A)Single measurement; ^(B)Mean of dual measurements; ^(C)Meanof six measurements

TABLE 7 shows the concentration of antiviral (μM) at which a confluentmonolayer of MDCK cells was protected over a 5 day period fromcytopathic viral infection.

Example 4 Cytoprotection Assays with Influenza B Strains

The candidate inhibitors were tested against Zanamivir for theircytopathic effect (CPE) against two influenza B strains. It is importantto note that, although the present Cytoprotection assay is a goodqualitative indicator of antiviral activity, the results are oftenvariable (as noted by others, Tisdale M. (2000)) and caution exercisedwhen doing a quantitative analysis.

TABLE 8 The activity of compounds against influenza B/HongKong/5/72Compound Cytoprotective effect (ng/mL) Relative activity Zanamivir 22191.0 4 (DFSA-4NH2) 1775.6 1.25 7 (N3-102ET) 3906.3 0.57 5 (N3-105ET)976.6 2.27 12 (DFSA-4Gu) 1775.6 1.25 13 (N3-106ET) 710.2 3.13 9(N3-111AMD) 15056.8 0.15 23DFSA 34090.9 0.07

TABLE 9 The activity of compounds against influenza B/Florida/04/06Replicate 1 Replicate 2 Cyto- Cyto- protective protective effectRelative effect Relative Compound (ng/mL) activity (ng/mL) activityZanamivir 2219 1.0 887 1.0 4 (DFSA-4NH2) 947 2.3 1074 0.83 7 (N3-102ET)3267 0.67 3267 0.27 5 (N3-105ET) 290 7.7 290 3.1

Example 5 In Vivo Pharmacokinetic (PK) Profile Study

The purpose of the study was to evaluate the pharmacokinetic profile ofa novel fluorosialic compound (DFSA-4Gu), in comparison with Zanamivirby the intranasal route and to compare the pharmacokinetic profile ofDFSA-4Gu when dosed by the intranasal and intravenous routes in theBalb/C mouse (Mus musculus).

TABLE 10 Study Parameters Gp Group # of Dose Admin. Dose Volume N per #Name Animals (mg/kg) Route (μL) time point Time-point 1 DFSA-IV 23 1Intravenous 200/20 g 4 5, 15, 30 min, 1, 2 hr (10 μL/g) 3 7 d 2 DFSA-IN23 1 Intranasal 10 μL/nostril/15 g 4 5, 15, 30 min, 1, 2 hr (0.67μL/nostril/g) 3 7 d 3 ZAN-IN 23 1 Intranasal 10 μL/nostril/15 g 4 5, 15,30 min, 1, 2 hr (0.67 μL/nostril/g) 3 7 d

A PK study was conducted to evaluate DFSA-4Gu in mice, administered byIN and IV routes, and to evaluate Zanamivir administered by the INroute, all at 1 mg/kg in a single dose. Tissue levels and plasma levelsfor DFSA-4Gu were obtained and lung tissue levels for Zanamivir wereobtained. Levels declined with apparent first order kinetics in alltissues by both routes. Comparison of DFSA-4Gu and Zanamivir by the INroutes showed that comparable compound exposure in the lung was observedwith both agents.

Calculated PK Parameters

PK parameters calculated for DFSA-4Gu and Zanamivir after intranasaldosing with a single 1 mg/kg dose.

TABLE 11A Calculated PK Values Tissue Compound AUC (ng × min/L) T_(1/2)(min) C_(initial) (ng/mL) Plasma DFSA-4Gu  76433 17.3 4954 TracheaDFSA-4Gu 421865 28.1 Lung DFSA-4Gu 236814 22.2 Lung Zanamivir 25408919.1PK parameters calculated for DFSA-4Gu after intravenous dosing with asingle 1 mg/kg dose.

TABLE 11B Calculated PK Values Tissue Compound AUC (ng × min/L) T_(1/2)(min) C_(initial) (ng/mL) Plasma DFSA-4Gu 88202  8.3 9480 TracheaDFSA-4Gu 24749  9.4 Lung DFSA-4Gu 25307 14.2

The apparent half-life for the compounds was longer after intranasaldosing than after intravenous dosing. As well the exposure, expressed asAUC for DFSA-4Gu was increased in trachea and lung by intranasal dosing,relative to intravenous dosing with AUC ratios (IN/IV) for lung=9.3 andfor trachea=17.0.

The AUC in lung after intranasal dosing of Zanamivir and DFSA-4Gu weresimilar, AUC(Lung) (Zanamivir/DFSA-4Gu)=1.07. The apparent half-lives ofthe two agents in the lung after intranasal dosing were also similar

Example 6 In Vivo Murine Model of Influenza Infection

The in vivo efficacy of Zanamivir, DFSA-4Gu & DFSA-4NH2 was tested in amouse model of influenza (HK1). Compounds were compared to Zanamavir,whereby animals received a single intranasal dose of drug prior toinfection and were given follow-up doses twice per day for a total ofsix days. Mortality due to infection was set at 15% weight loss afterwhich animals were euthanized. Survival of animals was significantlyextended in groups that received a 1 mg/Kg dose of DFSA-4GU andZanamavir, as compared to the untreated control group. Similarly,treatment with DFSA-4NH2 did somewhat extend survival as compared to theuntreated control group. However, drug treatments did not preventmortality as all treated animals eventually reached endpoint due toinfluenza infection. Although these results are promising, the use of adifferent virus strain or an increased dose of drugs may alter thesurvival.

TABLE 12 Experimental Groups and Treatments Gp Group Test Doses Virus #Name Article N (mg/kg/d) Strain 1 PR8-DFSA-4Gu DFSA-4Gu 10 1 A/Hong 2PR8-DFSA-4NH2 DFSA-4NH2 10 1 Kong/1/68 3 PR8-ZAN Zanamivir 10 0.1 (H3N2)4 PR8-ZAN Zanamivir 10 1 5 PR8-Untreated Saline 10 N/A 6DFSA-4NH2-Control DFSA-4NH2  5† 1 None 7 DFSA-4Gu-Control DFSA-4Gu  5 1Total N: 60 †The original protocol called for N = 3 for each healthycontrol group (Groups 6 and 7 in Table 6). In the execution of thestudy, N = 5 were used in these groups, increasing the total animalnumber to 60.

TABLE 13 Survival rates of animals on days 3, 4, 5, 6, 7 and 8 postinfection. TABLE 13 shows the number of animals remaining in each group.N = 10 for all groups except groups 6 and 7 (n = 5). Day Day Day Day DayDay Day Day Day Day Day 3 pm 4 am 4 pm 5 am 5 pm 6 am 6 pm 7 am 7 pm 8am 8 pm Group 1 HK1- 10 10 8 7 6 5 4 3 1 1 0 DFSA-Gu- (1 mg/kg) Group 2HK1- 10 10 3 2 1 0 0 0 0 0 0 DFSA-4NH2- (1 mg/kg) Group 3 HK1- 10 9 2 11 0 0 0 0 0 0 ZAN-(0.1 mg/kg) Group 4-HK1- 10 10 9 5 3 2 1 0 0 0 0ZAN-(1 mg/kg) Group 5 10 10 4 1 0 0 0 0 0 0 0 Untreated Group 6 DFSA- 55 5 5 5 5 5 5 5 5 5 4NH2-Control Group 7 DFSA- 5 5 5 5 5 5 5 5 5 5 54GU-Control

TABLE 14 Mean % weight loss per group on day 4 (am) post infection. Thistime point was selected because many animals were on the verge ofreaching endpoint, but most groups still had 10 animals. P- % weightloss value¹ mean +/− SD Group 1 HK1-DFSA-4Gu-1.0 0.01 10.75 +/− 4.87Group 2 HK1-DFSA-4N112-1.0 0.14 16.02 +/− 2.07 Group 3 HK1-ZAN-0.1 0.0616.73 +/− 2.62 Group 4-HK1-ZAN-1.0 0.01 12.24 +− 2.34 Group 6DFSA-4N112-Control 0.00  2.68 +/− 1.76 Group 7 DFSA-4GU-Control 0.00 4.72 +/− 2.78 Group 5 Untreated   15 +/− 2.06 ¹P-value determined bystudent's T-test in which each group was individually compared to themean of untreated group #7. A p-value below 0.05 was consideredstatistically significant.

With regards to the above results, and the potential alteration inprotocol, the use of a different virus strain, a different end point, oran increased dose of drugs may be justified. For example, another mouseadapted virus strain such as A/PR/8/34 may produce different results.Also, an endpoint of 15% weight loss may be too low, whereby animals maystill recover after losing 20% or more. For example Bantia et al. (2001)reported recovery of Zanamavir treated animals after more than 20percent weight loss. Similarly, a higher dose of drug may be required toachieve complete protection. For example, Levena et al. (2001), theauthors required doses of Zanamavir at 10 and 50 mg/kg/day before theyobserved a protective effect.

Fluorinated compounds of the class described in this invention areinhibitors of a range of glycosidases, and specific with respect totheir target enzymes. These compounds are mechanism-based in theirinhibitory action. They bind to the enzyme like the normal substrate andundergo the first step of catalysis (intermediate formation) like thenatural substrate, but then only very slowly undergo the second step(turnover via hydrolysis). Importantly, this mechanism-basis inhibitionshould make resistance formation by viruses more difficult. Since theinhibitors are mechanism-based, any mutations in the viral enzyme thatreduce the inhibition must necessarily reduce the efficiency of theenzyme on the natural substrate. FIG. 2 shows an example ofneuraminidase inhibition by 3-fluorosialyl fluoride (1, 23DFSA) (note:sialic acid numbering is different from that of normal sugars due to theanomeric carboxylate).

The fluorosialics differ fundamentally from Zanamivir and Oseltamivir intwo major ways. Zanamivir and Oseltamivir are reversibly bindinginhibitors that interact with the enzyme active site very tightly due totheir flattened, cyclic conformation. Their binding mode likely imitatesthe transition state conformation of the sugar during hydrolysis. Theyare therefore transition state mimics The fluorosialics, by contrast,contain no double bond thus adopt a regular chair conformation. Theyreact with the enzyme as if they are substrates and form a covalent bondwith the active site nucleophile, and only hydrolyse to products veryslowly. They derive their very high efficacies from the long-livednature of the intermediate formed.

Due to this completely different mode of action it was not evident thatincorporation of an amine or guanidine substituent would increase theireffectiveness since the sugar ring has a very different conformation inthe two cases: the amine/guanidine would be presented in a verydifferent manner in the two cases, thus likely interacts quitedifferently. Even more importantly, the major efficacy of thefluorosialics derives from the formation of a relatively long-livedcovalent intermediate. It was not at all evident that incorporation ofthe 4-amine/guanidine in place of the 4-hydroxyl would slow down thehydrolysis of this intermediate (deglycosylation) much more than itslowed the formation of the intermediate (glycosylation) with the resultof a much longer-lived intermediate thus a higher efficacy inhibitor.

Furthermore, comparative binding of these wild type and mutant virusesagainst Zanamivir, Oseltamivir, 4-G diF SA and 4-G3Feq diF SA wasconducted. Based on IC₅₀ kinetics and reactivation experimentspreviously conducted, it was known that these mutant viruses showedresistance to Zanamivir, but also to Peramivir and Oseltamivir, withrapid binding/dissociation of all.

TABLE 15 2,3-Fluorinated Glycosides having both a 3 equatorial FluorinesCompound Structure 4-G 3 F equatorial diF SA (4-G 3Feq diFSA) Compound4G3Feq

(alternative representation) 4-NH₂ 3 F equatorial diF SA (2F3F4NH₂)Compound 4NH₂3Feq

(alternative representation)

(alternative

Even though the tested viruses were the most resistant viruses handledto date, the viruses were surprisingly fit. Although the NA activity ofeach is very low, <10% of wild types, they still showed sufficient NA togrow to high titres in cell culture. Fitness in animals is yet to bedetermined.

Since some of the promising diF SA compounds have a 4-guanidino group itwas of interest whether the viruses would also show resistance to our4-guanidino compounds. Furthermore, this would potentially provide anindication of the relative roles of the formation of the covalentlinkage and the interactions with the guanidinium group in terms of highaffinity binding of the 4-G diF (control) and 4-G3Feq diF SA (test)compounds.

Example 7 Cytoprotection Assays with Various Viral Strains

TABLES 16-19 show the concentrations of control and test antivirals (μM)at which a confluent monolayer of MDCK cells was protected over a 5 dayperiod from cytopathic viral infection by a variety of viral strains.The test antiviral for the below tables is 4-G3Feq diF SA with a 3′equatorial F.

TABLE 16 Effects of mutations at E119A, D, K on sensitivity toinhibitors (IC₅₀ μM) Sol Isl Sol Isl Auckland Auckland B/FloridaB/Florida sH1N1 sH1N1 Fold pH1N1 pH1N1 nM wt E119D Fold Res wt E119A Reswt E119K Fold Res Zanamivir 3.9 >10,000 ≧10,000 2.2 >10,000 ≧10,0000.6 >100,000 ≧100,000 Oseltamivir 22.6 1205 53 1.0 419 419 0.2 147 6134-G diF SA 6.6 1515 229 181 >10,000 >1000 284 >100,000 >1000 4-G3Feq 2.027 13 4.7 130 27 4.0 >100,000 ≧10,000 diF SA

The results shown in TABLE 16 are consistent with previous testing ofother panels of viruses with 4-G diF SA. However, the 4-G3Feq SA has alower IC₅₀ than the 4-G diF SA for many of the resistant strains.

The E119K mutation appears to confer some resistance to both 4-G diF SAand 4-G3Feq diF SA, but less so to Oseltamivir (4-NH₂), suggesting thatthe interactions of the 4-guanidinium group are potentially significantto high affinity binding of not only the established NAIs but also toboth 4-G diF SAs. The most significant difference in resistance profileswas seen for the 4-G3Feq SA with the E119A mutation, thereis >10,000-fold resistance to the other 3 drugs, but only 27-fold to the4-G3Feq. Thus the 3F in the equatorial position results in significantlydifferent binding behaviour for the 4-G diF SA compared to the otherinhibitors with a 4-G group.

For the E119D there is lower resistance with the 4-G3Feq SA than the 4-GdiF SA, and importantly also several orders of magnitude lowerresistance than to Zanamivir. Since the E119D is significantly lessresistant to 4-G diFeq SA than to Zanamivir, this suggests theguanidinium in the fluorosialics is less likely to select for resistantstrains than that in zanamivir, likely due to the different (transientcovalent) mode of action.

TABLE 17 Effects of mutations at E119G, V on sensitivity to inhibitors(IC₅₀ μM) G70C G70C Fold Fukui Fukui Fold H1N9 H1N9 resis- H3N2 H3N2resis- wt E119G tance wt E119V tance Zanamivir 2.7 678 248 3.8 3.4 0.9Oseltamivir 2.8 2.9 1.1 1.7 260 155 4-G diF SA 66.7 1433 21 2006 998 0.54-G3Feq diF 136 17.3 0.1 24.9 2.4 0.1 SA

The E119G mutation appears to confer a high level resistance toZanamivir only, confers 20-fold resistance to 4G diF SA, whereas the4-G3Feq SA actually appears to bind better in the mutant than in thewild type.

The E119V mutation appears to confer a high level resistance toOseltamivir, but does not confer resistance to the 4G diF SA, Zanamivir,or 4-G3Feq diF SA.

TABLE 18 Effects of H274Y mutation on sensitivity to inhibitors (IC₅₀nM) Fold Fold sH1N1/01 sH1N/01 resis- sH1N1/08 sH1N1/08 resis- wt H274Ytance wt H274Y tance Zanamivir 1.9 2.2 1.2 1.0 2.0 2.0 Osel- 3.1 2440781 3.0 2000 667 tamivir 4-G diF 115.4 217 1.9 136 265 1.9 SA 4-G3Feq12.6 43.5 3.5 6.8 43.2 6.4 diF SA

4G diF SA or 4-G3Feq SA appear to still be effective against the H274Ymutation which confers high level Oseltamivir resistance, but not toZanamivir.

TABLE 19 Effects of D197E mutation on sensitivity to inhibitors (nM)B/Perth B/Perth wt D197E Fold resistance Zanamivir 8.9 257.5 28.9Oseltamivir 104.4 708.0 6.8 4-G diF SA 54.0 161.9 3.0 4-G3Feq diF SA 4.58.0 1.8

Mutations at D197 appear to confer cross resistance to known NAIs due toaltered interactions with the adjacent R152 and the N-acetyl group onthe ring. However, the 4-G3Feq SA does not appear to be affected by thisinteraction.

Example 8 Comparison of 3′ Axial with 3′ Equatorial Compounds on H1N1Viral Strains

TABLE 20 Comparison of 2 Equatorial/3 Axial and 2 Equatorial/3Equatorial Compounds with Both 4 Gu and 4 NH₂ in H1N1. H1N1 OsR H1N12F3F4Gu 2F3F4NH₂ 2F3F4Gu 2F3F4NH₂ 2eq3ax 2eq3eq 2eq3eq 2eq3ax 2eq3eq2eq3eq 2eq3ax ki (min⁻¹) 0.075 ± 0.002  1.5 ± 0.16 4.7 ± 0.1 0.094 ±0.002 2.0 ± 0.3 0.79 ± 0.27 0.50 ± 0.03 Ki (μM) 0.47 ± 0.06 0.50 ± 0.085.5 ± 0.2  0.25 ± 0.026 0.35 ± 0.19 0.23 ± 0.17 2.90 ± 0.72 ki/Ki(min⁻¹/ 0.16 3.0 0.85 0.37 5.8 3.5 0.18 μM) the ki/Ki

TABLE 21 Shows Reactivation Data for 2 Equatorial 3 Axial and 2Equatorial 3 Equatorial Compounds with Both 4 Gu and 4 NH₂. Half lifefor Strain Inactivator reactivation (hours) H1N1 OsR 2eq3ax4Gu 116 H1N1OsR 2eq3eq4Gu 9 H1N1 OsR 2eq3eq4NH2 2 H1N1 NML 2eq3eq4NH2 4

TABLE 20 shows formal kinetic data for inactivation of H1N1 and itsOseltamivir resistant mutant. The ki/Ki value is generally known as thespecificity parameter, thus is the most readily interpreted. For bothstrains the 2 equatorial/3 axial guanidine, the 2 equatorial/3equatorial-fluoro guanidine, and the 2 equatorial/3 equatorial-fluoroamine derivatives were tested, but the the 2 equatorial/3 axial-fluoroamine derivative was only tested against H1N1 and not against theOseltamivir resistant mutant. Interestingly, the 3Feq 4-amineinactivates H1N1 approximately 20 fold faster than does the 3Fax4-amine, which is approximately the same ratio as we see for the 3Fequatorial versus the 3 axial guanidines on both strains. Accordingly,inactivation by the 3Feq 4-amine is faster than by the 3Fax 4-guanidine.One possible concern was that reactivation (turnover) of theintermediate formed by the 3-F-equatorial compounds would be too fast tobe useful. However, while TABLE 21 shows that reactivation is indeedfaster, the half lives of 2-4 hours are expected to be sufficient forthese compounds to function well in vivo.

As seen above, the 3′ equatorial compounds are better at maintainingpotency against some viral strains than the 3′ axial counterparts.Furthermore, various resistant strains of virus appear to remainsensitive to the 3′ equatorial compounds even when sensitivitydiminishes for the 3′ axial compounds. In addition, the amine isgenerally expected to have better oral bioavailability than theguanidine.

The enzyme IC₅₀ kinetics, reactivation, plaque reduction assays andcross-resistance data all suggest the 4-G3Feq diF SA is a superiorinhibitor to the 4-G diF SA. Resistance data also supports the 4-G3FeqSA as having a different resistance profile than Zanamivir, Oseltamivir,and 4-G diF SA. Thus the 3′ equatorial F leads to novel interactions ofthe 4-G group at the ground and transition states, thus avoidingresistance seen with many mutations at E119.

Fluorinated compounds of the class described herein are inhibitors of arange of glycosidases, and specific with respect to their targetenzymes. These compounds are mechanism-based in their inhibitory action.They bind to the enzyme like the normal substrate and undergo the firststep of catalysis (intermediate formation) like the natural substrate,but then only very slowly undergo the second step (turnover viahydrolysis). Importantly, this mechanism-basis inhibition should makeresistance formation by viruses more difficult. Since the inhibitors aremechanism-based, any mutations in the viral enzyme that reduce theinhibition must necessarily reduce the efficiency of the enzyme on thenatural substrate. Please note that the sialic acid numbering isdifferent from that of aldose-sugars due to the anomeric carboxylate.

The fluorosialics described herein differ fundamentally from Zanamivirand Oseltamivir in two major ways. Zanamivir and Oseltamivir arereversibly binding inhibitors that interact with the enzyme active sitevery tightly due to their flattened, cyclic conformation. Their bindingmode likely imitates the transition state conformation of the sugarduring hydrolysis. They are therefore transition state mimics. Thefluorosialics described herein, by contrast, contain no double bond thusadopt a regular chair conformation. They react with the enzyme as ifthey are substrates and form a covalent bond with the active sitenucleophile, and only hydrolyse to products very slowly. They derivetheir very high efficacies primarily from the long-lived nature of theintermediate formed.

It was not evident that a 3′ equatorial F substituent would increase theeffectiveness of these compounds against resistant viral strains strainsas compared to stereoisomers having 3′ axial F configuration.

Although various embodiments of the invention are disclosed herein, manyadaptations and modifications may be made within the scope of theinvention in accordance with the common general knowledge of thoseskilled in this art. Such modifications include the substitution ofknown equivalents for any aspect of the invention in order to achievethe same result in substantially the same way. Numeric ranges areinclusive of the numbers defining the range. The word “comprising” isused herein as an open-ended term, substantially equivalent to thephrase “including, but not limited to”, and the word “comprises” has acorresponding meaning. As used herein, the singular forms “a”, “an” and“the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a thing” includes more thanone such thing. Citation of references herein is not an admission thatsuch references are prior art to the present invention.

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1-42. (canceled)
 43. A method of modulating viral neuraminidase activitycomprising contacting a viral neuraminidase with a compound selectedfrom the group consisting of:

or a pharmaceutically acceptable salt thereof.
 44. The method accordingto claim 43, wherein the viral neuraminidase is a GH34 neuraminidase.45. The method according to claim 43, wherein the viral neuraminidase isan influenza neuraminidase.
 46. The method according to claim 45,wherein modulating a viral neuraminidase comprises administering thecompound or pharmaceutically acceptable salt thereof to an animal havinginfluenza, and is effective to treat influenza in the animal.
 47. Themethod according to claim 46, wherein the animal is a mammal.
 48. Themethod according to claim 46, wherein the animal is a human.
 49. Amethod of treating influenza in an animal comprising administering tothe animal a compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof, wherein the compoundmodulates viral neuraminidase activity.
 50. The method according toclaim 49, wherein the viral neuraminidase is a GH34 neuraminidase. 51.The method according to claim 49, wherein the viral neuraminidase is aninfluenza neuraminidase.
 52. The method according to claim 49, whereinthe animal is a mammal.
 53. The method according to claim 49, whereinthe animal is a human.
 54. A pharmaceutical composition comprising apharmaceutically acceptable carrier or excipient and a compound selectedfrom the group consisting of:

or a pharmaceutically acceptable salt thereof.